U.S. patent application number 11/257286 was filed with the patent office on 2006-02-23 for epitope testing using soluble hla.
Invention is credited to Rico Buchli, Heather D. Hickman, William H. Hildebrand, Kiley R. Prilliman.
Application Number | 20060040310 11/257286 |
Document ID | / |
Family ID | 27487068 |
Filed Date | 2006-02-23 |
United States Patent
Application |
20060040310 |
Kind Code |
A1 |
Hildebrand; William H. ; et
al. |
February 23, 2006 |
Epitope testing using soluble HLA
Abstract
The present invention relates generally to a methodology for
assaying the binding of a peptide to an individual, specific,
soluble HLA molecule. The peptides utilized in the method may be
identified by indirect methods utilizing T lymphocytes, or by a
direct method of epitope discovery described herein.
Inventors: |
Hildebrand; William H.;
(Edmond, OK) ; Buchli; Rico; (Edmomd, OK) ;
Prilliman; Kiley R.; (San Diego, CA) ; Hickman;
Heather D.; (Oklahoma City, OK) |
Correspondence
Address: |
DUNLAP, CODDING & ROGERS P.C.
PO BOX 16370
OKLAHOMA CITY
OK
73113
US
|
Family ID: |
27487068 |
Appl. No.: |
11/257286 |
Filed: |
October 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10095818 |
Mar 11, 2002 |
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11257286 |
Oct 24, 2005 |
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09974366 |
Oct 10, 2001 |
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11257286 |
Oct 24, 2005 |
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10022066 |
Dec 18, 2001 |
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11257286 |
Oct 24, 2005 |
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60274605 |
Mar 9, 2001 |
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60362799 |
Mar 7, 2002 |
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Current U.S.
Class: |
435/6.14 ;
435/7.1 |
Current CPC
Class: |
A61K 39/385 20130101;
A61K 2039/55555 20130101; A61K 9/1272 20130101; C07K 14/70571
20130101; G01N 33/6878 20130101; C07K 14/4702 20130101; A61K
2039/622 20130101; G01N 33/5008 20130101; C12N 2740/16122 20130101;
C07K 2319/00 20130101; C12N 9/6421 20130101; C12N 9/1247 20130101;
C07K 14/4728 20130101; C07K 14/47 20130101; A61K 2039/605 20130101;
G01N 33/5041 20130101; C07K 14/70539 20130101; C07K 14/78 20130101;
C07K 14/005 20130101; A61K 39/39 20130101 |
Class at
Publication: |
435/006 ;
435/007.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method of assaying a peptide for binding to an individual
class I molecule, the method comprising the steps of: providing a
peptide of interest; providing individual soluble class I
molecule-endogenous peptide complexes in which individual soluble
class I molecules have endogenous peptides loaded therein; mixing
the peptide of interest with the individual soluble class I
molecules; and identifying individual soluble class I
molecule-peptide of interest complexes, wherein the individual
soluble class I molecules have the peptide of interest loaded
therein.
2. The method of claim 1 further comprising the step of treating
the individual soluble class I molecule-endogenous peptide
complexes under conditions that cause the individual soluble class
I molecules to release the endogenous peptides prior to mixing the
peptide of interest with the individual soluble class I
molecules.
3. The method of claim 2 wherein the step of treating the
individual soluble class I molecules-endogenous peptide complexes
involves heating the individual soluble class I molecule-endogenous
peptide complexes to cause the individual soluble class I molecules
to release the endogenous peptides.
4. The method of claim 1 wherein, in the steps of providing the
peptide of interest and identifying individual soluble class I
molecule-peptide of interest complexes, the peptide of interest is
labeled to allow identification of individual soluble class I
molecule-peptide of interest complexes from unbound peptide of
interest.
5. The method of claim 4 wherein the peptide of interest is labeled
with a radiolabel or a fluorescent label.
6. The method of claim 5 wherein, in the step of identifying
individual soluble class I molecule-peptide of interest complexes,
the peptide of interest is labeled with a fluorescent label, and
the individual soluble class I molecule-peptide of interest
complexes are identified by fluorescence polarization.
7. The method of claim 1 wherein, in the method of providing
individual soluble class I molecule-endogenous peptide complexes,
the individual soluble class I molecule-endogenous peptide
complexes are produced by the method comprising the steps of:
obtaining genomic DNA or cDNA encoding at least one class I
molecule; identifying an allele encoding an individual class I
molecule in the genomic DNA or cDNA; PCR amplifying the allele
encoding the individual class I molecule in a locus specific manner
such that a PCR product produced therefrom encodes a truncated,
soluble form of the individual class I molecule; cloning the PCR
product into an expression vector, thereby forming a construct that
encodes the individual soluble class I molecule; transfecting the
construct into a cell line to provide a cell line containing a
construct that encodes an individual soluble class I molecule, the
cell line being able to naturally process proteins into peptide
ligands capable of being loaded into antigen binding grooves of
class I molecules; culturing the cell line under conditions which
allow for expression of the individual soluble class I molecules
from the construct, such conditions also allowing for endogenous
loading of a peptide ligand into the antigen binding groove of each
individual soluble class I molecule prior to secretion of the
individual soluble class I molecules from the cell; and isolating
the secreted individual soluble class I molecules having the
endogenously loaded peptide ligands bound thereto.
8. The method of claim 7 wherein the construct further encodes a
tag which is attached to the individual soluble class I molecule
and aids in isolating the individual soluble class I molecule.
9. The method of claim 8 wherein the tag is selected from the group
consisting of a HIS tail and a FLAG tail.
10. The method of claim 1 wherein, in the step of providing a
peptide of interest, the peptide of interest is identified by a
method for identifying at least one endogenously loaded peptide
ligand that distinguishes an infected cell from an uninfected cell,
the method comprising the steps of: providing an uninfected cell
line containing a construct that encodes an individual soluble
class I molecule, the uninfected cell line being able to naturally
process proteins into peptide ligands capable of being loaded into
antigen binding grooves of class I molecules; infecting a portion
of the uninfected cell line with at least one of a microorganism, a
gene from a microorganism or a tumor gene, thereby providing an
infected cell line; culturing the uninfected cell line and the
infected cell line under conditions which allow for expression of
individual soluble class I molecules from the construct, such
conditions also allowing for endogenous loading of a peptide ligand
in the antigen binding groove of each individual soluble class I
molecule prior to secretion of the individual soluble class I
molecules from the cell; isolating the secreted individual soluble
class I molecules having the endogenously loaded peptide ligands
bound thereto from the uninfected cell line and the infected cell
line; separating the endogenously loaded peptide ligands from the
individual soluble class I molecules from the uninfected cell line
and separating the endogenously loaded peptide ligands from the
individual soluble class I molecules from the infected cell line;
isolating the endogenously loaded peptide ligands from the
uninfected cell line and the endogenously loaded peptide ligands
from the infected cell line; comparing the endogenously loaded
peptide ligands isolated from the infected cell line to the
endogenously loaded peptide ligands isolated from the uninfected
cell line; and identifying at least one endogenously loaded peptide
ligand presented by the individual soluble class I molecule on the
infected cell line that is not presented by the individual soluble
class I molecule on the uninfected cell line.
11. The method of claim 10 further comprising the step of
identifying a source protein from which the at least one
endogenously loaded peptide ligand presented by the individual
soluble class I molecule on the infected cell line and not
presented by the individual soluble class I molecule on the
uninfected cell line is obtained.
12. The method of claim 10 wherein, in the step of identifying at
least one endogenously loaded peptide ligand presented by the
individual soluble class I molecule on the infected cell line but
not on the uninfected cell line, the at least one endogenously
loaded peptide ligand is obtained from a protein encoded by at
least one of the microorganism, the gene from a microorganism or
the tumor gene with which the cell line was infected to form the
infected cell line.
13. The method of claim 10 wherein, in the step of identifying at
least one endogenously loaded peptide ligand presented by the
individual soluble class I molecule on the infected cell line but
not on the uninfected cell line, the at least one endogenously
loaded peptide ligand is obtained from a protein encoded by the
uninfected cell line.
14. The method of claim 13, wherein the protein encoded by the
uninfected cell line from which the at least one endogenously
loaded peptide ligand is obtained has increased expression in a
tumor cell line.
15. The method of claim 10 wherein, in the step of infecting a
portion of the uninfected cell line, the portion of the uninfected
cell line is infected with HIV.
16. The method of claim 1 wherein, in the step of providing a
peptide of interest, the peptide of interest is identified by a
method for identifying at least one endogenously loaded peptide
ligand that distinguishes an infected cell from an uninfected cell,
the method comprising the steps of: providing an uninfected cell
line containing a construct that encodes an individual soluble
class I molecule, the uninfected cell line being able to naturally
process proteins into peptide ligands capable of being loaded into
antigen binding grooves of class I molecules; infecting a portion
of the uninfected cell line with at least one of a microorganism, a
gene from a microorganism or a tumor gene, thereby providing an
infected cell line; culturing the uninfected cell line and the
infected cell line under conditions which allow for expression of
the individual soluble class I molecules from the construct, such
conditions also allowing for endogenous loading of a peptide ligand
into the antigen binding groove of each individual soluble class I
molecule prior to secretion of the individual soluble class I
molecules from the cell; isolating the secreted individual soluble
class I molecules having the endogenously loaded peptide ligands
bound thereto from the uninfected cell line and the infected cell
line; separating the endogenously loaded peptide ligands from the
individual soluble class I molecules from the uninfected cell line
and separating the endogenously loaded peptide ligands from the
individual soluble class I molecules from the infected cell line;
isolating the endogenously loaded peptide ligands from the
uninfected cell line and the endogenously loaded peptide ligands
from the infected cell line; comparing the endogenously loaded
peptide ligands isolated from the uninfected cell line to the
endogenously loaded peptide ligands isolated from the infected cell
line; and identifying at least one endogenously loaded peptide
ligand presented by the individual soluble class I molecule on the
uninfected cell line that is not presented by the individual
soluble class I molecule on the infected cell line.
17. The method of claim 16 further comprising the step of
identifying a source protein from which the at least one
endogenously loaded peptide ligand presented by the individual
soluble class I molecule on the uninfected cell line and not
presented by the individual soluble class I molecule on the
infected cell line is obtained.
18. The method of claim 17 wherein, in the step of infecting a
portion of the uninfected cell line, the portion of the uninfected
cell line is infected with HIV.
19. The method of claim 1 wherein, in the step of identifying
individual soluble class I molecule peptide of interest complexes,
the complexes are identified using an antibody that recognizes the
individual soluble class I molecule having a peptide loaded
therein.
20. A method of assaying a peptide for binding to an individual
class I molecule, the method comprising the steps of: providing a
peptide of interest; labeling the peptide of interest; providing
individual soluble class I molecules; mixing the labeled peptide of
interest with the individual soluble class I molecules; and
identifying individual soluble class I molecule-labeled peptide of
interest complexes, wherein the individual soluble class I
molecules have the labeled peptide of interest loaded therein.
21. The method of claim 20 wherein, in the step of labeling the
peptide of interest, the peptide of interest is labeled with a
radiolabel or a fluorescent label.
22. The method of claim 21 wherein, in the steps of labeling the
peptide of interest and identifying individual soluble class I
molecule-labeled peptide of interest complexes, the peptide of
interest is labeled with a fluorescent label, and the individual
soluble class I molecule-labeled peptide of interest complexes are
identified by fluorescence polarization.
23. The method of claim 21 wherein, in the steps of labeling the
peptide of interest and identifying individual soluble class I
molecule-labeled peptide of interest complexes, the peptide of
interest is labeled with a radiolabel, and the individual soluble
class I molecule-radiolabeled peptide of interest complexes are
isolated from unbound radiolabeled peptide of interest.
24. The method of claim 23 wherein the individual soluble class I
molecule-radiolabeled peptide of interest complexes are isolated
from unbound radiolabeled peptide of interest by gel filtration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/095,818, filed Mar. 11, 2002, now abandoned; this application
also claims priority under 35 U.S.C. .sctn. 119(e) of provisional
U.S. Ser. No. 60/274,605, filed Mar. 9, 2001, entitled "HLA PROTEIN
PRODUCTION FROM CDNA", and provisional U.S. Ser. No. 60/362,799,
filed Mar. 7, 2002, entitled "EPITOPE TESTING USING SOLUBLE HLA",
the contents of which are hereby expressly incorporated in their
entirety by reference. This application is also a
continuation-in-part of U.S. Ser. No. 09/974,366, filed Oct. 10,
2001, entitled "COMPARATIVE LIGAND MAPPING FROM MHC CLASS I
POSITIVE CELLS;" and is also a continuation-in-part of U.S. Ser.
No. 10/022,066, filed Dec. 18, 2001, entitled "METHOD AND APPARATUS
FOR THE PRODUCTION OF SOLUBLE MHC ANTIGENS AND USES THEREOF," the
contents of which are also hereby expressly incorporated in their
entirety by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to a methodology of
epitope testing for the identification of peptides that bind to an
individual soluble MHC Class I or Class II molecule.
[0005] 2. Description of the Background Art
[0006] Class I major histocompatibility complex (MHC) molecules,
designated HLA class I in humans, bind and display peptide antigen
ligands upon the cell surface. The peptide antigen ligands
presented by the class I MHC molecule are derived from either
normal endogenous proteins ("self") or foreign proteins ("nonself")
introduced into the cell. Nonself proteins may be products of
malignant transformation or intracellular pathogens such as
viruses. In this manner, class I MHC molecules convey information
regarding the internal fitness of a cell to immune effector cells
including but not limited to, CD8.sup.+ cytotoxic T lymphocytes
(CTLs), which are activated upon interaction with "nonself"
peptides, thereby lysing or killing the cell presenting such
"nonself" peptides.
[0007] Class II MHC molecules, designated HLA class II in humans,
also bind and display peptide antigen ligands upon the cell
surface. Unlike class I MHC molecules which are expressed on
virtually all nucleated cells, class II MHC molecules are normally
confined to specialized cells, such as B lymphocytes, macrophages,
dendritic cells, and other antigen presenting cells which take up
foreign antigens from the extracellular fluid via an endocytic
pathway. The peptides they bind and present are derived from
extracellular foreign antigens, such as products of bacteria that
multiply outside of cells, wherein such products include protein
toxins secreted by the bacteria that often times have deleterious
and even lethal effects on the host (e.g., human). In this manner,
class II molecules convey information regarding the fitness of the
extracellular space in the vicinity of the cell displaying the
class II molecule to immune effector cells, including but not
limited to, CD4.sup.+ helper T cells, thereby helping to eliminate
such pathogens the examination of such pathogens is accomplished by
both helping B cells make antibodies against microbes, as well as
toxins produced by such microbes, and by activating macrophages to
destroy ingested microbes.
[0008] Class I and class II HLA molecules exhibit extensive
polymorphism generated by systematic recombinatorial and point
mutation events; as such, hundreds of different HLA types exist
throughout the worlds's population, resulting in a large
immunological diversity. Such extensive HLA diversity throughout
the population results in tissue or organ transplant rejection
between individuals as well as differing susceptibilities and/or
resistances to infectious diseases. HLA molecules also contribute
significantly to autoimmunity and cancer. Because HLA molecules
mediate most, if not all, adaptive immune responses, large
quantities of pure isolated HLA proteins are required in order to
effectively study transplantation, autoimmunity disorders, and for
vaccine development.
[0009] There are several applications in which purified, individual
class I and class II MHC proteins are highly useful. Such
applications include using MHC-peptide multimers as
immunodiagnostic reagents for disease resistance/autoimmunity;
assessing the binding of potentially therapeutic peptides; elution
of peptides from MHC molecules to identify vaccine candidates;
screening transplant patients for preformed MHC specific
antibodies; and removal of anti-HLA antibodies from a patient.
Since every individual has differing MHC molecules, the testing of
numerous individual MHC molecules is a prerequisite for
understanding the differences in disease susceptibility between
individuals. Therefore, purified MHC molecules representative of
the hundreds of different HLA types existing throughout the world's
population are highly desirable for unraveling disease
susceptibilities and resistances, as well as for designing
therapeutics such as vaccines.
[0010] Class I HLA molecules alert the immune response to disorders
within host cells. Peptides, which are derived from viral- and
tumor-specific proteins within the cell, are loaded into the class
I molecule's antigen binding groove in the endoplasmic reticulum of
the cell and subsequently carried to the cell surface. Once the
class I HLA molecule and its loaded peptide ligand are on the cell
surface, the class I molecule and its peptide ligand are accessible
to cytotoxic T lymphocytes (CTL). CTL survey the peptides presented
by the class I molecule and destroy those cells harboring ligands
derived from infectious or neoplastic agents within that cell.
[0011] While specific CTL targets have been identified, little is
known about the breadth and nature of ligands presented on the
surface of a diseased cell. From a basic science perspective, many
outstanding questions have permeated through the art regarding
peptide exhibition. For instance, it has been demonstrated that a
virus can preferentially block expression of HLA class I molecules
from a given locus while leaving expression at other loci intact.
Similarly, there are numerous reports of cancerous cells that fail
to express class I HLA at particular loci. However, there is no
data describing how (or if) the three classical HLA class I loci
differ in the immunoregulatory ligands they bind. It is therefore
unclear how class I molecules from the different loci vary in their
interaction with viral- and tumor-derived ligands and the number of
peptides each will present.
[0012] Discerning virus- and tumor-specific ligands for CTL
recognition is an important component of vaccine design. Ligands
unique to tumorigenic or infected cells can be tested and
incorporated into vaccines designed to evoke a protective CTL
response. Several methodologies are currently employed to identify
potentially protective peptide ligands. One approach uses T cell
lines or clones to screen for biologically active ligands among
chromatographic fractions of eluted peptides (Cox et al., Science,
vol 264, 1994, pages 716-719, which is expressly incorporated
herein by reference in its entirety). This approach has been
employed to identify peptides ligands specific to cancerous cells.
A second technique utilizes predictive algorithms to identify
peptides capable of binding to a particular class I molecule based
upon previously determined motif and/or individual ligand sequences
(De Groot et al., Emerging Infectious Diseases, (7) 4, 2001, which
is expressly incorporated herein by reference in its entirety).
Peptides having high predicted probability of binding from a
pathogen of interest can then be synthesized and tested for T cell
reactivity in precursor, tetramer or ELISpot assays.
[0013] However, there has been no readily available source of
individual HLA molecules. The quantities of HLA protein available
have been small and typically consist of a mixture of different HLA
molecules. Production of HLA molecules traditionally involves
growth and lysis of cells expressing multiple HLA molecules. Ninety
percent of the population is heterozygous at each of the HLA loci;
codominant expression results in multiple HLA proteins expressed at
each HLA locus. To purify native class I or class II molecules from
mammalian cells requires time-consuming and cumbersome purification
methods, and since each cell typically expresses multiple
surface-bound HLA class I or class II molecules, HLA purification
results in a mixture of many different HLA class I or class II
molecules. When performing experiments using such a mixture of HLA
molecules or performing experiments using a cell having multiple
surface-bound HLA molecules, interpretation of results cannot
directly distinguish between the different HLA molecules, and one
cannot be certain that any particular HLA molecule is responsible
for a given result. Therefore, prior to the present invention, a
need existed in the art for a method of producing substantial
quantities of individual HLA class I or class II molecules so that
they can be readily purified and isolated independent of other HLA
class I or class II molecules. Such individual HLA molecules, when
provided in sufficient quantity and purity as described herein,
provides a powerful tool for studying and measuring immune
responses.
[0014] Therefore, there exists a need in the art for improved
methods of assaying binding of peptides to class I and class II MHC
molecules to identify epitopes that bind to specific individual
class I and class II MHC molecules. The present invention solves
this need by coupling the production of soluble HLA molecules with
epitope isolation, discovery, and testing methodology.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. Overview of 2 stage PCR strategy to amplify a
truncated version of the human class I MHC.
[0016] FIG. 2. Flow chart of the epitope discovery of
C-terminal-tagged sHLA molecules. Class I positive transfectants
are infected with a pathogen of choice and sHLA preferentially
purified utilizing the tag. Subtractive comparison of MS ion maps
yields ions present only in infected cell, which are then MS/MS
sequenced to derive class I epitopes.
[0017] FIG. 3. A*0201T saturation curve of increasing amount of
peptide/sHLA complex loaded on a coated W6/32 ELISA plate.
[0018] FIG. 4. Comparison of saturation curves created using either
sHLA allele (A*0201T) or detergent lysate A*0201 obtained through
cell extraction demonstrating higher sensitivity of sHLA than
detergent lysate.
[0019] FIG. 5. Diagrammatic scheme of the peptide exchange reaction
using Fluorescence Polarization.
[0020] FIG. 6. Binding of P2 to sHLA at room temperature showing
net increase after t=0 subtraction.
[0021] FIG. 7. Binding of P2 to sHLA at 4.degree. C. (preincubated
at 54.degree. C.) showing net increase after t=0 subtraction.
[0022] FIG. 8. Binding of various peptides (P1-P5) to sHLA A*0201T
over a range of concentration detected by Fluorescence Polarization
at time 45 hours after mixing.
[0023] FIG. 9. Binding of various peptides (P1-P5) to SHLA A*0201T
using Fluorescence Polarization at a concentration of 50 .mu.g/ml
sHLA.
[0024] FIG. 10. Competition assay for sHLA A*0201T loaded with the
specific standard peptide P5(FITC).
DETAILED DESCRIPTION OF THE INVENTION
[0025] Before explaining at least one embodiment of the invention
in detail by way of exemplary drawings, experimentation, results,
and laboratory procedures, it is to be understood that the
invention is not limited in its application to the details of
construction and the arrangement of the components set forth in the
following description or illustrated in the drawings,
experimentation and/or results. The invention is capable of other
embodiments or of being practiced or carried out in various ways.
As such, the language used herein is intended to be given the
broadest possible scope and meaning; and the embodiments are meant
to be exemplary--not exhaustive. Also, it is to be understood that
the phraseology and terminology employed herein is for the purpose
of description and should not be regarded as limiting.
[0026] The present invention combines methodologies for assaying
the binding of peptide epitopes to individual, soluble MHC
molecules with methodologies for the production of individual,
soluble MHC molecules and with a method of epitope discovery and
comparative ligand mapping (including methods of distinguishing
infected/tumor cells from uninfected/non-tumor cells). The method
of production of individual, soluble MHC molecules has previously
been described in detail in parent application U.S. Ser. No.
10/022,066, filed Dec. 18, 2001, entitled "METHOD AND APPARATUS FOR
THE PRODUCTION OF SOLUBLE MHC ANTIGENS AND USES THEREOF," the
contents of which have previously been expressly incorporated in
their entirety by reference. The method of epitope discovery and
comparative ligand mapping has previously been described in detail
in parent application U.S. Ser. No. 09/974,366, filed Oct. 10,
2001, entitled "COMPARATIVE LIGAND MAPPING FROM MHC CLASS I
POSITIVE CELLS", the contents of which have previously been
expressly incorporated in their entirety by reference. A brief
description of each of these methodologies is included herein below
for the purpose of exemplification and should not be considered as
limiting.
[0027] The present invention is related to a method of assaying a
peptide for binding to an individual class I molecule. The method
includes providing a peptide of interest and providing individual
soluble class I molecule-endogenous peptide complexes in which
individual soluble class I molecules have endogenous peptides
loaded therein. The peptide of interest and the individual soluble
class I molecules are mixed together, and individual soluble class
I molecule-peptide of interest complexes, wherein the individual
soluble class I molecules have the peptide of interest loaded
therein, are identified. The individual soluble class I
molecule-endogenous peptide complexes may be treated under
conditions that cause the individual soluble class I molecules to
release the endogenous peptides prior to mixing the peptide of
interest with the individual soluble class I molecules, and such
method of treating may involve heating the individual soluble class
I molecule-endogenous peptide complexes to cause the individual
soluble class I molecules to release the endogenous peptides.
[0028] The peptide of interest may be labeled using a radiolabel or
a fluorescent label to allow identification of individual soluble
class I molecule-peptide of interest complexes from unbound peptide
of interest. When a fluorescent label is utilized, the individual
soluble class I molecule-peptide of interest complexes may be
identified by any of the methods of fluorescence detection known in
the art, such as by fluorescence polarization. Alternatively, the
individual soluble class I molecule-fluorescence labeled peptide of
interest may be isolated using an antibody against the individual
soluble class I molecule. When a radiolabel is utilized, the
individual soluble class I molecule-peptide of interest complexes
may be isolated away from unbound peptide of interest, such as by
gel filtration. Alternatively, the individual soluble class I
molecule-peptide of interest complexes may be identified using an
antibody that recognizes the individual soluble class I molecule
having a peptide loaded therein.
[0029] To produce the individual soluble class I
molecule-endogenous peptide complexes, genomic DNA or cDNA encoding
at least one class I molecule is obtained, and an allele encoding
an individual class I molecule in the genomic DNA or cDNA is
identified. The allele encoding the individual class I molecule is
PCR amplified in a locus specific manner such that a PCR product
produced therefrom encodes a truncated, soluble form of the
individual class I molecule. The PCR product is then cloned into an
expression vector, thereby forming a construct that encodes the
individual soluble class I molecule, and the construct is
transfected into a cell line to provide a cell line containing a
construct that encodes an individual soluble class I molecule. The
cell line must be able to naturally process proteins into peptide
ligands capable of being loaded into antigen binding grooves of
class I molecules.
[0030] The cell line is then cultured under conditions which allow
for expression of the individual soluble class I molecules from the
construct, and these conditions also allow for endogenous loading
of a peptide ligand into the antigen binding groove of each
individual soluble class I molecule prior to secretion of the
individual soluble class I molecules from the cell. The secreted
individual soluble class I molecules having the endogenously loaded
peptide ligands bound thereto are then isolated.
[0031] The construct that encodes the individual soluble class I
molecule may further encode a tag, such as a HIS tail or a FLAG
tail, which is attached to the individual soluble class I molecule
and aids in isolating the individual soluble class I molecule.
[0032] The peptide of interest may be chosen based on several
methods of epitope discovery known in the art. Alternatively, the
peptide of interest may be identified by a method for identifying
at least one endogenously loaded peptide ligand that distinguishes
an infected cell from an uninfected cell. Such method includes
providing an uninfected cell line containing a construct that
encodes an individual soluble class I molecule, wherein the
uninfected cell line is able to naturally process proteins into
peptide ligands capable of being loaded into antigen binding
grooves of class I molecules. A portion of the uninfected cell line
is infected with at least one of a microorganism (such as HIV or
HBV), a gene from a microorganism or a tumor gene, thereby
providing an infected cell line, and both the uninfected cell line
and the infected cell line are cultured under conditions which
allow for expression of individual soluble class I molecules from
the construct. The culture conditions also allow for endogenous
loading of a peptide ligand in the antigen binding groove of each
individual soluble class I molecule prior to secretion of the
individual soluble class I molecules from the cell. The secreted
individual soluble class I molecules having the endogenously loaded
peptide ligands bound thereto are isolated from the uninfected cell
line and the infected cell line, and the endogenously loaded
peptide ligands are separated from the individual soluble class I
molecules from both the uninfected cell line and the infected cell
line. The endogenously loaded peptide ligands are then isolated
from both the uninfected cell line and the infected cell line, and
the two sets of endogenously loaded peptide ligands are compared to
identify at least one endogenously loaded peptide ligand presented
by the individual soluble class I molecule on the infected cell
line that is not presented by the individual soluble class I
molecule on the uninfected cell line, or to identify at least one
endogenously loaded peptide ligand presented by the individual
soluble class I molecule on the uninfected cell line that is not
presented by the individual soluble class I molecule on the
infected cell line.
[0033] Following identification of the peptide ligand that
distinguishes an infected cell from an uninfected cell, a source
protein from which the endogenously loaded peptide ligand is
obtained can be identified. Such source protein may be encoded by
at least one of the microorganism, the gene from a microorganism or
the tumor gene with which the cell line was infected to form the
infected cell line, or the source protein may be encoded by the
uninfected cell line. When the source protein is encoded by the
uninfected cell line, such protein may also demonstrate increased
expression in a tumor cell line.
Production of Individual, Soluble MHC Molecules
[0034] The methods of the present invention may, in one embodiment,
utilize a method of producing MHC molecules (from genomic DNA or
cDNA) that are secreted from mammalian cells in a bioreactor unit.
Substantial quantities of individual MHC molecules are obtained by
modifying class I or class II MHC molecules so that they are
capable of being secreted, isolated, and purified. Secretion of
soluble MHC molecules overcomes the disadvantages and defects of
the prior art in relation to the quantity and purity of MHC
molecules produced. Problems of quantity are overcome because the
cells producing the MHC do not need to be detergent lysed or killed
in order to obtain the MHC molecule. In this way the cells
producing secreted MHC remain alive and therefore continue to
produce MHC. Problems of purity are overcome because the only MHC
molecule secreted from the cell is the one that has specifically
been constructed to be secreted. Thus, transfection of vectors
encoding such secreted MHC molecules into cells which may express
endogenous, surface bound MHC provides a method of obtaining a
highly concentrated form of the transfected MHC molecule as it is
secreted from the cells. Greater purity is assured by transfecting
the secreted MHC molecule into MHC deficient cell lines.
[0035] Production of the MHC molecules in a hollow fiber bioreactor
unit allows cells to be cultured at a density substantially greater
than conventional liquid phase tissue culture permits. Dense
culturing of cells secreting MHC molecules further amplifies the
ability to continuously harvest the transfected MHC molecules.
Dense bioreactor cultures of MHC secreting cell lines allow for
high concentrations of individual MHC proteins to be obtained.
Highly concentrated individual MHC proteins provide an advantage in
that most downstream protein purification strategies perform better
as the concentration of the protein to be purified increases. Thus,
the culturing of MHC secreting cells in bioreactors allows for a
continuous production of individual MHC proteins in a concentrated
form.
[0036] The method of producing MHC molecules utilized in the
present invention and described in detail in U.S. Ser. No.
10/022,066 begins by obtaining genomic or complementary DNA which
encodes the desired MHC class I or class II molecule. Alleles at
the locus which encode the desired MHC molecule are PCR amplified
in a locus specific manner. These locus specific PCR products may
include the entire coding region of the MHC molecule or a portion
thereof. In one embodiment a nested or hemi-nested PCR is applied
to produce a truncated form of the class I or class II gene so that
it will be secreted rather than anchored to the cell surface. FIG.
1 illustrates the PCR products resulting from such nested PCR
reactions. In another embodiment the PCR will directly truncate the
MHC molecule.
[0037] Locus specific PCR products are cloned into a mammalian
expression vector and screened with a variety of methods to
identify a clone encoding the desired MHC molecule. The cloned MHC
molecules are DNA sequenced to ensure fidelity of the PCR. Faithful
truncated clones of the desired MHC molecule are then transfected
into a mammalian cell line. When such cell line is transfected with
a vector encoding a recombinant class I molecule, such cell line
may either lack endogenous class I MHC molecule expression or
express endogenous class I MHC molecules. One of ordinary skill of
the art would note the importance, given the present invention,
that cells expressing endogenous class I MHC molecules may
spontaneously release MHC into solution upon natural cell death. In
cases where this small amount of spontaneously released MHC is a
concern, the transfected class I MHC molecule can be "tagged" such
that it can be specifically purified away from spontaneously
released endogenous class I molecules in cells that express class I
molecules. For example, a DNA fragment encoding a HIS tail may be
attached to the protein by the PCR reaction or may be encoded by
the vector into which the PCR fragment is cloned, and such HIS
tail, therefore, further aids in the purification of the class I
MHC molecules away from endogenous class I molecules. Tags beside a
histidine tail have also been demonstrated to work, and one of
ordinary skill in the art of tagging proteins for downstream
purification would appreciate and know how to tag a MHC molecule in
such a manner so as to increase the ease by which the MHC molecule
may be purified.
[0038] Cloned genomic DNA fragments contain both exons and introns
as well as other non-translated regions at the 5' and 3' termini of
the gene. Following transfection into a cell line which transcribes
the genomic DNA (gDNA) into RNA, cloned genomic DNA results in a
protein product thereby removing introns and splicing the RNA to
form messenger RNA (mRNA), which is then translated into an MHC
protein. Transfection of MHC molecules encoded by gDNA therefore
facilitates reisolation of the gDNA, mRNA/cDNA, and protein.
Production of MHC molecules in non-mammalian cell lines such as
insect and bacterial cells requires cDNA clones, as these lower
cell types do not have the ability to splice introns out of RNA
transcribed from a gDNA clone. In these instances the mammalian
gDNA transfectants of the present invention provide a valuable
source of RNA which can be reverse transcribed to form MHC cDNA.
The cDNA can then be cloned, transferred into cells, and then
translated into protein. In addition to producing secreted MHC,
such gDNA transfectants therefore provide a ready source of mRNA,
and therefore cDNA clones, which can then be transfected into
non-mammalian cells for production of MHC. Thus, the present
invention which starts with MHC genomic DNA clones allows for the
production of MHC in cells from various species.
[0039] A key advantage of starting from gDNA is that viable cells
containing the MHC molecule of interest are not needed. Since all
individuals in the population have a different MHC repertoire, one
would need to search more than 500,000 individuals to find someone
with the same MHC complement as a desired individual--such a
practical example of this principle is observed when trying to find
a donor to match a recipient for bone marrow transplantation. Thus,
if it is desired to produce a particular MHC molecule for use in an
experiment or diagnostic, a person or cell expressing the MHC
allele of interest would first need to be identified.
Alternatively, in the method of the present invention, only a
saliva sample, a hair root, an old freezer sample, or less than a
milliliter (0.2 ml) of blood would be required to isolate the gDNA.
Then, starting from gDNA, the MHC molecule of interest could be
obtained via a gDNA clone as described herein, and following
transfection of such clone into mammalian cells, the desired
protein could be produced directly in mammalian cells or from cDNA
in several species of cells using the methods of the present
invention described herein.
[0040] Current experiments to obtain an MHC allele for protein
expression typically start from mRNA, which requires a fresh sample
of mammalian cells that express the MHC molecule of interest.
Working from gDNA does not require gene expression or a fresh
biological sample. It is also important to note that RNA is
inherently unstable and is not as easily obtained as is gDNA.
Therefore, if production of a particular MHC molecule starting from
a cDNA clone is desired, a person or cell line that is expressing
the allele of interest must traditionally first be identified in
order to obtain RNA. Then a fresh sample of blood or cells must be
obtained; experiments using the methodology of the present
invention show that .gtoreq.5 milliliters of blood that is less
than 3 days old is required to obtain sufficient RNA for MHC cDNA
synthesis. Thus, by starting with gDNA, the breadth of MHC
molecules that can be readily produced is expanded. This is a key
factor in a system as polymorphic as the MHC system; hundreds of
MHC molecules exist, and not all MHC molecules are readily
available. This is especially true of MHC molecules unique to
isolated populations or of MHC molecules unique to ethnic
minorities. Starting class I or class II MHC molecule expression
from the point of genomic DNA simplifies the isolation of the gene
of interest and insures a more equitable means of producing MHC
molecules for study; otherwise, one would be left to determine
whose MHC molecules are chosen and not chosen for study, as well as
to determine which ethnic population from which fresh samples
cannot be obtained and therefore should not have their MHC
molecules included in a diagnostic assay.
[0041] While cDNA may be substituted for genomic DNA as the
starting material, production of cDNA for each of the desired HLA
class I types will require hundreds of different, HLA typed, viable
cell lines, each expressing a different HLA class I type.
Alternatively, fresh samples are required from individuals with the
various desired MHC types. The use of genomic DNA as the starting
material allows for the production of clones for many HLA molecules
from a single genomic DNA sequence, as the amplification process
can be manipulated to mimic recombinatorial and gene conversion
events. Several mutagenesis strategies exist whereby a given class
I gDNA clone could be modified at either the level of gDNA or at
the cDNA resulting from this gDNA clone. The process of producing
MHC molecules utilized in the present invention does not require
viable cells, and therefore the degradation which plagues RNA is
not a problem.
Method of Epitope Testing
[0042] Utilizing the production of individual soluble MHC proteins
described herein previously, a method of epitope testing has been
developed. Such a method of epitope testing is advantageous because
of the single specificities of the soluble HLA molecules produced
in the manner described herein. Previous work in this area has used
mixtures of different HLA molecules to purify or separate out
individual class I or class II HLA molecules from the mixture.
However, such purification steps result in selective purification
of particular endogenously loaded peptide/HLA complexes while not
purifying the same HLA molecule complexed with a different peptide.
Such selective purification will bias the endogenously loaded
peptides in the HLA molecules. For example, typical purification
methods involve the use of antibodies, which may recognize certain
peptides loaded in HLA molecules and not others. In addition, such
purification steps may not remove all of the other class I and
class II HLA molecules, leaving these proteins to skew data and its
interpretation. Therefore, the antibody may not recognize all of a
particular class I or class II HLA because of the peptide bound
therein. Using individual, soluble HLA molecules produced in
accordance with the present invention in the methods of epitope
testing described herein, the individual, soluble HLA molecules are
endogenously loaded with thousands of naturally produced peptides,
and the pool of such individual, soluble HLA molecules is not
biased in the peptide cargo loaded therein.
[0043] The goal of the epitope binding assay is to identify the
affinity of peptide ligands for binding to particular HLA
molecules. The initial HLA binding studies utilized detergent
solubilized class I molecules from EBV transformed cell lines
(Sette, A., et al., Mol Immunol, 31(11):813-22 (1994), which is
hereby expressly incorporated herein by reference). One perceived
advantage of utilizing HLA that is naturally loaded with thousands
of endogenous peptide ligands inside the cell is that peptide
binding competition assays actually utilize these thousands of
naturally loaded peptides in the assay. In the competitive assay
with naturally loaded HLA, the HLA molecule of interest can be
purified away from other HLA molecules in the detergent lysate or
be used in a mixture with other HLA molecules. Radiolabeled
peptides are identified that have a high affinity for the HLA
molecule in question. The affinity of additional "test" peptides
for the HLA molecule in question is then determined by their
ability to compete with the high affinity radiolabeled peptide.
[0044] Several challenges are associated with the isolation of HLA
with naturally loaded peptides. One challenge is that most EBV cell
lines express 6 different class I molecules. Purification of HLA
molecules can result in the isolation of class I containing only a
subset of the representative peptide ligands (Solheim, J. C., et
al., J. Immunol., 151(10):5387-5397 (1993); and Bluestone, J. A.,
et al., Journal of Exp. Med., 176(6):1757-61 (1992), each of which
are hereby expressly incorporated herein by reference).
Alternatively, using a mixture of HLA molecules produces results
where the investigator does not know which HLA molecule in the
mixture actually bound the peptide. A third challenge is that
isolation of HLA proteins from detergent cell lysates produces
microgram quantities of peptide. When performing a peptide binding
assay twice in triplicate with hundreds to thousands of peptides at
several different concentrations for each peptide, HLA protein can
be rapidly utilized in milligram quantities.
[0045] To circumvent this limitation in the amount of HLA protein
available with naturally loaded ligands, two groups have reported
producing HLA proteins in bacteria (Ostergaard Pedersen, L., et
al., Eur J Immunol, 31(10):2986-96 (2001); and Dedier, S., et al.,
J Immunol Methods, 255(1-2):57-66 (2001), each of which are
expressly incorporated herein by reference). The HLA produced in
bacteria must then be refolded into a natural conformation
consisting of class I heavy chain, beta-2-microglobulin light
chain, and peptide ligand(s). While this method of class I protein
production circumvents the limitations of detergent lysate HLA
protein production, production in bacteria (or insect cells)
introduces another set of challenges to obtaining HLA protein for a
peptide binding study.
[0046] One challenge of producing non-native HLA heavy chains and
then refolding in vitro is that refolding in vitro can be
difficult. Discussions with Dr. John Altman at Emory University,
The Beckman Tetramer Facility, and others who work with bacterial
HLA indicates that the percentage of HLA that refolds into an
intact trimolecular complex of heavy chain-light chain-peptide can
range between 0 to 70%. Some HLA molecules come back together well
while others do not. One factor in refolding HLA molecules in vitro
is the peptide; high affinity peptides help to reform the complex
while some peptides result in no HLA refolding. Using those that do
refold with a high affinity peptide in a peptide binding assay can
be difficult because displacing a single high affinity peptide can
be difficult in the binding assay. This is in contrast to the
various affinities of the thousands of endogenously loaded
peptides. On the other hand, some HLA molecules cannot be refolded
when made in bacteria. Finally, although much HLA heavy chain can
be made, sometimes only a fraction of the protein will refold.
[0047] The method of producing sHLA with thousands of naturally
loaded endogenous peptides as disclosed herein above allows for the
production of milligram quantities of HLA in a natural form.
Peptides produced in this manner have been characterized and shown
to be the same as detergent solubilized HLA (Prilliman, K. R., et
al, Immunogenetics, 45:379-385 (1997); Prilliman, K. R., et al.,
Immunogenetics, 48:89-97 (1998); Prilliman, K. R., et al., Tissue
Antigens, 54(5):450-60 (1999); and Prilliman, K. R., et al., J
Immunol, 162(12):7277-84 (1999), each of which are expressly
incorporated herein by reference). By secreting HLA proteins we
insure that only the desired HLA molecule is in solution. Such HLA
molecules can then be purified without biasing the peptides in the
HLA protein. It has also been shown that high-affinity peptides
known to bind a particular HLA specificity bind the HLA molecule in
question, while peptides known to bind other HLA molecules do not
bind the HLA molecule in question. Additionally, it has been shown
that we can label the high affinity peptides with fluorescent
labels and then perform a competition assay that demonstrates that
other peptides can compete with the high affinity labeled peptide
at increasing concentrations. We therefore have produced
substantial quantities of a sHLA reagent that is natural in its
conformation and peptide cargo and that is useful for peptide
binding assays. The plentiful amount of protein allows the
screening of hundreds or thousands of peptides at different
concentrations in multiple experiments. The naturally loaded
peptide provides a true competitive reflection of the affinity of
the labeled and test peptides for the HLA molecule in question.
[0048] In one embodiment of the method of epitope testing,
purified, individual, soluble HLA molecules containing endogenously
loaded peptide ligands are mixed with a control peptide known to
have high affinity for the HLA molecule and the peptide(s) of
interest. A method of detection for binding of the known high
affinity control peptide is provided, such as labeling of the
control peptide, i.e., fluorescence or radiolabeling, or providing
an antibody specific for the complex formed when the known high
affinity control peptide binds to the individual HLA molecule
(FIGS. 3 and 4). The detectable, high affinity control peptide will
displace the endogenously loaded peptide ligands and bind to the
individual soluble HLA molecule, as detected by measurement of the
label on the control peptide or antibody binding to the control
peptide/HLA complex, and the peptide(s) of interest will compete
with the high affinity control peptides for displacement of the
endogenously loaded peptides and binding to the individual soluble
HLA molecules. A negative control used in such method would be a
low affinity peptide which will not compete with the high affinity
control peptide for displacement of the endogenously loaded peptide
and binding to the individual soluble HLA molecules. Thus, epitope
testing utilizing the soluble HLA molecules of the present
invention, simply put, involves a competition between the control
peptide and the test peptide by determining how well did the test
peptide displace the control peptide from a soluble HLA molecule
and/or displace an endogenously loaded peptide and competitively
bind to a soluble HLA molecule.
[0049] The above-described assay will typically be performed in
liquid phase; however, the method of epitope testing described
herein is adaptable to solid phase assays. That is, the individual
HLA molecules may be bound to a support. Methods of binding
proteins to a support are known in the art and are adaptable to the
assay methods described herein.
[0050] In yet another embodiment of a method of epitope testing
using the individual soluble HLA molecules described herein, a
number of individual, soluble HLA molecules may be bound to
Luminex.TM. beads. In the Luminex.TM. technology, a series of 100
to 1000 beads are each provided with varying concentrations of two
different dyes. For example, Bead A may comprise 10% Dye A and 90%
Dye B, while Bead B comprises 15% Dye A and 85% Dye B, while Bead C
comprises 20% Dye A and 80% Dye B, and Bead D comprises 25% Dye A
and 75% Dye B, etc. A specific fluorescence detector can identify
each specific bead by the amounts of Dye A and Dye B present in
each bead. To each of the 100 to 1000 Luminex.TM. beads, a
different individual class I or II HLA molecule can be bound. It
has been demonstrated that the individual soluble HLA molecules of
the present invention can be bound to the Luminex.TM. beads (U.S.
Ser. No. 60/274,605, which has previously been incorporated herein
by reference). Therefore, the fluorescence detector can identify
specific individual HLA molecules by the specific amount of dyes in
the bead to which such molecules are bound. All of the beads may be
mixed together in one assay, such as one well on a 96 well plate.
To the reaction, a detectable, high affinity control peptide as
described above is added for each individual soluble HLA molecule
bound to a bead, and the peptide of interest is also added to the
reaction. The mixture is then passed through the fluorescence
detector, which contains two different lasers. The first laser
detects the amounts of dyes in the bead, that is, the first laser
identifies which bead is passing the laser. The second laser is
positioned so that upon identification of the specific bead passing
the first laser, the second laser can detect whether or not the
detectable high affinity control peptide is bound to the HLA
molecule attached to the specific bead. As stated above, this
detection may be a radiolabel or fluorescence label attached to the
control peptide or a method of antibody binding which detects the
control peptide/HLA complex. In this manner, 100 to 1000 binding
tests can be run for each peptide of interest to determine which
HLA molecules bind such peptide.
[0051] In another embodiment of the method of epitope testing, as
HLA molecules are known to bind 9 amino acid peptides in the groove
thereof, nonamer peptides may be tethered to a chip or other type
of support to provide a peptide microarray. For example, PVDF
membranes have been utilized to prepare protein microarrays, and
such technology could be easily adaptable to the methods described
herein. Such nonamers may consist of every possible combination of
amino acids, and therefore 9.sup.20 combinations of nonamers would
be utilized, or such nonamers may only differ at positions known to
be important for binding to HLA molecules, for example, positions 2
and 9 (in this case, 2.sup.20 combinations would be required). The
nonamers may be anchored to the chip or other type of support in
any known manner. Preferably, the nonamers are anchored internally
off side chains of amino acids not on the ends of the nonamers.
Specific individual class I or class II HLA molecules could then be
passed over the nonamer microarray and allowed to bind, and by
detecting binding of specific class I or class II HLA molecules to
certain peptides, a database of all peptides to which individual
class I or class II HLA molecules bind could be formed (as
described in U.S. Ser. No. 10/082,034, filed Feb. 21, 2002,
entitled "SOLUBLE HLA LIGAND DATABASE UTILIZING PREDICTIVE
ALGORITHMS AND METHODS OF MAKING AND USING SAME", the contents of
which are hereby expressly incorporated in their entirety by
reference). In this manner, rather than making all the different
combinations of peptides produced from a specific virus, bacteria,
tumor gene, etc. to determine which peptides bound to individual
class I or class II MHC molecules, the genomic sequence of such
virus, bacteria, etc. could be "blasted" against the data obtained
above and located in the database to identify putative epitopes
which could be utilized in the method of epitope testing described
herein.
[0052] In another embodiment of the method of epitope testing, the
above described technology could be "flipped", that is, the
individual HLA molecules could be bound to the chip or other
support to form an HLA protein microarray. Such method would be
similar to the solid-phase assays described herein before.
Peptides Utilized in Epitope Testing
[0053] The peptides to be tested in the epitope binding assay of
the present invention will be synthetic peptides and will have
their sequence derived from portions of various host, viral and
bacterial proteins. The proteins upon which the peptides are based
can be identified in several ways. One method of identifying
proteins that contain peptides that may or may not bind to HLA
molecules is through the use of T lymphocytes. T lymphocytes are
known to recognize specific peptides in the context of specific HLA
molecules. Indeed, this is the mechanism whereby T lymphocytes
specifically target antigens in adaptive immune responses.
[0054] There are several means of using T lymphocytes for
identifying HLA holding peptides that trigger the T lymphocyte. One
means is to separate the complex mixture of peptides eluted from
HLA molecules into fractions or points in time. The peptides in the
fractions are then loaded onto HLA molecules and the cells with the
loaded peptides are exposed to T lymphocytes from a cancer patient,
a virus infected person, or a person with a bacterial infection.
The idea is that the T lymphocytes from a person with a disease
controlled by T lymphocytes can be used to identify T lymphocyte
peptide-HLA immune targets.
[0055] There are several ways to measure the recognition of
HLA-peptide target cells using T lymphocytes. One method is to
detect HLA/peptide target cell lysis by T lymphocytes, as disclosed
in Smith, E. S., et al., Lethality-based selection of recombinant
genes in mammalian cells: application to identifying tumor
antigens. Nat Med, 2001. 7(8): p. 967-72; Huczko, E. L., et al.,
Characteristics of endogenous peptides eluted from the class I MHC
molecule HLA-B7 determined by mass spectrometry and computer
modeling. J. Immunol., 1993. 151: p. 2572-2587; Scheibenbogen, C.,
et al., Analysis of the T cell response to tumor and viral peptide
antigens by an IFNg-ELISPOT assay. Int. J. Cancer, 1997. 71: p.
932-936; and Rinaldo, C. R., Jr., et al., Anti-human
immunodefciency virus type 1 (HIV-1) CD8(+) T-lymphocyte reactivity
during combination antiretroviral therapy in HIV-1-infected
patients with advanced immunodeficiency. J Virol, 2000. 74(9): p.
4127-38, all of which are incorporated herein expressly by
reference. Using one or a combination of assays that detect T
lymphocyte recognition of a target cell, experiments can be
designed to identify HLA-peptide candidates that may trigger T
lymphocytes.
[0056] In the above references, investigators using T lymphocytes
to identify HLA-peptide targets do not know which peptide is
stimulating T lymphocytes. For example, Zauderer et al. know that
over-expression of a transfected "tumor" gene in a cell line is
causing T lymphocytes from cancer patients to recognize that cell
line. However, this assay cannot determine which peptide from the
protein encoded by the transfected "tumor" gene is triggering T
lymphocyte recognition. Moreover, the investigator does not know if
a peptide derived from the protein encoded by the "tumor" gene is
presented by HLA or if the gene is indirectly changing other
proteins which may indirectly result in another protein's peptide
being recognized in HLA.
[0057] Other uses of T lymphocytes indicate that HLA-peptide target
complexes exist, but again, they do not specify which exact target
peptide is in the HLA molecule. Using evidence from the T
lymphocyte assay, an investigator will synthesize several peptides
which are suspected to resemble the T lymphocyte target. These
peptides can then be tested for their binding to HLA target
molecules. Peptides that bind to the HLA molecules are detected in
this binding assay. Such binding to an HLA molecule indicates that
a peptide should next be tested for recognition by T lymphocytes.
Peptides that do not bind can be eliminated from further
characterization.
[0058] Use of T lymphocytes is one way to begin to sift through
proteins and peptide mixtures for possible immune targets. Another
way is to simply synthesize all the overlapping peptides in a
target protein. These overlapping peptides can be tested for their
binding to HLA proteins. Peptides that bind can then be tested for
T lymphocyte recognition. This process eliminates T lymphocytes
from the selection process.
[0059] A protein that an investigator wishes to target in a
vaccine, diagnostic, or therapy can be selected in a number of
ways. These protein selection mechanisms include T lymphocyte
selection, microarray identification of upregulated genes, or
simply testing immune responses to a protein of interest. These
selection criteria are then partnered to an epitope binding assay
as described herein to identify portions of a protein that bind
well to one or many HLA proteins. Another method that can be
coupled to the epitope binding test described here is a predictive
database. A predictive database can indicate which peptides in
which proteins might bind to HLA, and then the HLA binding test
described herein can confirm or deny these predictions.
[0060] The above methods for identifying epitopes for the peptide
binding assay are indirect methods. That is, the peptides that may
be immunogenic are not directly identified as immunogenic. As such,
the epitope testing assay described herein confirms which peptides
actually do bind HLA molecules. The epitope testing assay also
determines how well a peptide binds and to which HLA molecule(s)
the peptide binds. Determining to which HLA molecule(s) a peptide
binds is important because the population is heterogeneous for HLA.
Therefore, a peptide which binds many HLA is a good vaccine
candidate because a vaccine incorporating that peptide will work in
a broader range of the population. The peptide binding assay
therefore indicates how many HLA and how well to each HLA a peptide
epitope binds.
[0061] The peptide binding assay can also be coupled with direct
epitope discovery methods. For example, peptide epitopes unique to
infected and cancerous cells can be directly identified by
producing sHLA molecules in cancerous and infected cells and then
sequencing the epitopes unique to the cancerous or infected cells.
Such epitopes can then be tested for their binding to various HLA
molecules to see how many HLA molecules these epitopes might bind.
This direct method of epitope discovery is described in detail in
U.S. Ser. No. 09/974,366 and is briefly described hereinbelow.
[0062] In summary, a number of methods can be used to select
peptides which may bind to HLA and may be immune targets. The
epitope binding assay can determine whether the putative peptide
target actually binds to a specific HLA molecule and how well this
epitope binds the specific HLA in comparison to other known
peptides that bind to the specific HLA. The epitope binding assay
can also be used to screen panels of overlapping synthetic peptides
to help sift through large numbers of potential vaccine/diagnostic
candidates. Those peptides that bind well can then be tested for
immunogenicity. Finally, the epitope binding assay can determine if
a particular peptide binds multiple HLA molecules as well as the
peptide's relative affinity for various HLA molecules.
Methods of Epitope Discovery and Comparative Ligand Mapping
[0063] As stated above, peptides utilized in the peptide binding
assay can be identified by direct epitope discovery methods. For
example, peptide epitopes unique to infected and cancerous cells
can be directly identified by producing sHLA molecules in cancerous
and infected cells and then sequencing the epitopes unique to the
cancerous or infected cells. Such epitopes can then be tested for
their binding to various HLA molecules to see how many HLA
molecules these epitopes might bind. This direct method of epitope
discovery is described in detail in U.S. Ser. No. 09/974,366 and is
briefly described hereinbelow.
[0064] The method of epitope discovery included in the present
invention (and described in detail in U.S. Ser. No. 09/974,366)
includes the following steps: (1) providing a cell line containing
a construct that encodes an individual soluble class I or class II
MHC molecule (wherein the cell line is capable of naturally
processing self or nonself proteins into peptide ligands capable of
being loaded into the antigen binding grooves of the class I or
class II MHC molecules); (2) culturing the cell line under
conditions which allow for expression of the individual soluble
class I or class II MHC molecule from the construct, with such
conditions also allowing for the endogenous loading of a peptide
ligand (from the self or non-self processed protein) into the
antigen binding groove of each individual soluble class I or class
II MHC molecule prior to secretion of the soluble class I or class
II MHC molecules having the peptide ligands bound thereto; and (4)
separating the peptide ligands from the individual soluble class I
or class II MHC molecules.
[0065] Class I and class II MHC molecules are really a trimolecular
complex consisting of an alpha chain, a beta chain, and the
alpha/beta chain's peptide cargo (i.e., peptide ligand) which is
presented on the cell surface to immune effector cells. Since it is
the peptide cargo, and not the MHC alpha and beta chains, which
marks a cell as infected, tumorigenic, or diseased, there is a
great need to identify and characterize the peptide ligands bound
by particular MHC molecules. For example, characterization of such
peptide ligands greatly aids in determining how the peptides
presented by a person with MHC-associated diabetes differ from the
peptides presented by the MHC molecules associated with resistance
to diabetes. As stated above, having a sufficient supply of an
individual MHC molecule, and therefore that MHC molecule's bound
peptides, provides a means for studying such diseases. Because the
method of the present invention provides quantities of MHC protein
previously unobtainable, unparalleled studies of MHC molecules and
their important peptide cargo can now be facilitated and utilized
to distinguish infected/tumor cells from uninfected/non-tumor cells
by unique epitopes presented by MHC molecules in the disease or
non-disease state.
[0066] The method of the present invention includes the direct
comparative analysis of peptide ligands eluted from class I HLA
molecules (as described previously in U.S. Ser. No. 09/974,366).
The disclosure of U.S. Ser. No. 09/974,366 demonstrates that the
addition of a C-terminal epitope tag (such as a 6-HIS or FLAG tail)
to transfected class I molecules has no effects on peptide binding
specificity of the class I molecule and consequently has no
deleterious effects on direct peptide ligand mapping and
sequencing, and also does not disrupt endogenous peptide
loading.
[0067] The method described in parent application U.S. Ser. No.
09/974,366 further relates to a novel method for detecting those
peptide epitopes which distinguish the infected/tumor cell from the
uninfected/non-tumor cell. The results obtained from the present
inventive methodology cannot be predicted or ascertained
indirectly; only with a direct epitope discovery method can the
unique epitopes described therein be identified. Furthermore, only
with this direct approach can it be ascertained that the source
protein is degraded into potentially immunogenic peptide epitopes.
Finally, this unique approach provides a glimpse of which proteins
are uniquely up and down regulated in infected/tumor cells.
[0068] The utility of such HLA-presented peptide epitopes which
mark the infected/tumor cell are three-fold. First, diagnostics
designed to detect a disease state (i.e., infection or cancer) can
use epitopes unique to infected/tumor cells to ascertain the
presence/absence of a tumor/virus. Second, epitopes unique to
infected/tumor cells represent vaccine candidates. Here, we
describe epitopes which arise on the surface of cells infected with
HIV. Such epitopes could not be predicted without natural virus
infection and direct epitope discovery. The epitopes detected are
derived from proteins unique to virus infected and tumor cells.
These epitopes can be used for virus/tumor vaccine development and
virus/tumor diagnostics. Third, the process indicates that
particular proteins unique to virus infected cells are found in
compartments of the host cell they would otherwise not be found in.
Thus, we identify uniquely upregulated or trafficked host proteins
for drug targeting to kill infected cells.
[0069] The parent application U.S. Ser. No. 09/974,366 describes,
in particular, peptide epitopes unique to HIV infected cells.
Peptide epitopes unique to the HLA molecules of HIV infected cells
were identified by direct comparison to HLA peptide epitopes from
uninfected cells by the method illustrated in the flow chart of
FIG. 2. Such method has been shown to be capable of identifying:
(1) HLA presented peptide epitopes, derived from intracellular host
proteins, that are unique to infected cells but not found on
uninfected cells, and (2) that the intracellular source-proteins of
the peptides are uniquely expressed/processed in HIV infected cells
such that peptide fragments of the proteins can be presented by HLA
on infected cells but not on uninfected cells.
[0070] The method of epitope discovery and comparative ligand
mapping also, therefore, describes the unique expression of
proteins in infected cells or, alternatively, the unique
trafficking and processing of normally expressed host proteins such
that peptide fragments thereof are presented by HLA molecules on
infected cells. These HLA presented peptide fragments of
intracellular proteins represent powerful alternatives for
diagnosing virus infected cells and for targeting infected cells
for destruction (i.e., vaccine development).
[0071] A group of the host source-proteins for HLA presented
peptide epitopes unique to HIV infected cells represent
source-proteins that are uniquely expressed in cancerous cells. For
example, through using the methodology of the present invention a
peptide fragment of reticulocalbin is uniquely found on HIV
infected cells. A literature search indicates that the
reticulocalbin gene is uniquely upregulated in cancer cells (breast
cancer, liver cancer, colorectal cancer). Thus, the HLA presented
peptide fragment of reticulocalbin which distinguishes HIV infected
cells from uninfected cells can be inferred to also differentiate
tumor cells from healthy non-tumor cells. Thus, HLA presented
peptide fragments of host genes and gene products that distinguish
the tumor cell and virus infected cell from healthy cells have been
directly identified. The epitope discovery method is also capable
of identifying host proteins that are uniquely expressed or
uniquely processed on virus infected or tumor cells. HLA presented
peptide fragments of such uniquely expressed or uniquely processed
proteins can be used as vaccine epitopes and as diagnostic
tools.
[0072] The methodology to target and detect virus infected cells
may not be to target the virus-derived peptides. Rather, the
methodology of the present invention indicates that the way to
distinguish infected cells from healthy cells is through
alterations in host encoded protein expression and processing. This
is true for cancer as well as for virus infected cells. The
methodology according to the present invention results in data
which indicates without reservation that proteins/peptides
distinguish virus/tumor cells from healthy cells.
[0073] In a brief example of the methodology of comparative ligand
mapping utilized in the methods of the present invention, a cell
line producing individual, soluble MHC molecules is constructed as
described hereinbefore and in U.S. Ser. No. 10/022,066. A portion
of the transfected cell line is cocultured with a virus of
interest, resulting in high-titre virus and providing infected
cells. In the example provided herein and in detail in U.S. Ser.
No. 10/022,066, the virus of interest is HIV. Alternatively, a
portion of the cell line producing individual, soluble MHC
molecules may be transformed to produce a tumor cell line.
[0074] The non-infected cell line and the cell line infected with
HIV are both cultured in hollow-fiber bioreactors as described
hereinabove and in detail in U.S. Ser. No. 10/022,066, and the
soluble HLA-containing supernatant is then removed from the
hollow-fiber bioreactors. The uninfected and infected harvested
supernatants were then treated in an identical manner post-removal
from the cell-pharm.
[0075] MHC class I-peptide complexes were affinity purified from
the infected and uninfected supernatants using W6/32 antibody.
Following elution, peptides were isolated from the class I
molecules and separated by reverse phase HPLC fractionation.
Separate but identical (down to the same buffer preparations)
peptide purifications were done for each peptide-batch from
uninfected and infected cells.
[0076] Fractionated peptides were then mapped by mass spectrometry
to generate fraction-based ion maps. Spectra from the same fraction
in uninfected/infected cells were manually aligned and visually
assessed for the presence of differences in the ions represented by
the spectra. Ions corresponding to the following categories were
selected for MS/MS sequencing: (1) upregulation in infected cells
(at least 1.5 fold over the same ion in uninfected cells), (2)
downregulation in infected cells (at least 1.5 fold over the same
ion in the uninfected cells), (3) presence of the ion only in
infected cells, or (4) absence of ion in infected cells that is
present in uninfected cells. In addition, multiple parameters were
established before peptides were assigned to one of the above
categories, including checking the peptide fractions preceding and
following the peptide fraction by MS/MS to ensure that the peptide
of interest was not present in an earlier or later fraction as well
as generation of synthetic peptides and subjection to MS/MS to
check for an exact match. In addition, one early quality control
step involves examining the peptide's sequence to see if it fits
the "predicted motif" defined by sequences that were previously
shown to be presented by the MHC molecule utilized.
[0077] After identification of the epitopes, literature searches
were performed on source proteins to determine their function
within the infected cell, and the source proteins were classified
into groups according to functions inside the cell. Secondly,
source proteins were scanned for other possible epitopes which may
be bound by other MHC class I alleles. Peptide binding predictions
were employed to determine if other peptides presented from the
source proteins were predicted to bind, and proteasomal prediction
algorithms were likewise employed to determine the likelihood of a
peptide being created by the proteasome.
[0078] In accordance with the present invention, Table I lists
peptide ligands that have been identified as being presented by the
B*0702 class I MHC molecule in cells infected with the HIV MN-1
virus but not in uninfected cells, and also lists one peptide
ligand that has been identified as not being presented by the
B*0702 class I MHC molecule in cells infected with the HIV MN-1
virus that is presented in uninfected cells. As one of ordinary
skill in the art can appreciate the novelty and usefulness of the
present methodology in directly identifying such peptide ligands
and the importance such identification has for numerous therapeutic
(vaccine development, drug targeting) and diagnostic tools.
[0079] As stated above, Table I identifies the sequences of peptide
ligands identified to date as being unique to HIV infected cells.
Class I sHLA B*0702 TABLE-US-00001 TABLE I OBS'D SEQ ION FRACTION
SEQUENCE MW MW SOURCE PROTEIN ID NO: Peptides Identified on
Infected cells that are not present on Uninfected Cells 612.720
32INF EQMFEDIISL 1223.582 1223.418 HIV MN-1, ENV 1 509.680 31INF
IPCLLISFL 1017.601 1017.334 CHOLINERGIC 2 RECEPTOR, ALPHA-3
POLYPEPTIDE 469.180 31INF STTAICATGL 936.466 936.360
UBIQUITIN-SPECIFIC 3 PROTEASE 420.130 16INF APAQNPEL 838.426
838.259 B-ASSOCIATED 4 TRANSCRIPT PROTEIN 3 (BAT3) 500.190 28INF
LVMAPRTVL 998.602 998.396 HLA-B HEAVY CHAIN 5 LEADER SEQUENCE
529.680 31INF APR[NS] 1057.388 UNKNOWN, CLOSE TO 6 PADX SEVERAL
cDNA's 523.166 12INF TPQSNRPVm 1044.500 1044.333 RNA POLYMERASE II
7 POLYPEPTIDE A 444.140 16INF AARPATSTL 887.495 887.280 EUK,
TRANSLATION 8 INITIATION FACTOR 4 470.650 16INF MAMMAALMA 940.413
939.410 SPARC-LIKE PROTEIN 9 490.620 16INF IATVDSYVI 979.240
TENASCIN-C 10 (HEXABRACHION) 563.640 16INF SPNQARAQAAL 1126.597
1126.364 POLYPYRIMIDINE 11 TRACT-BINDING PROTEIN 1 30INF GPRTAALGLL
968.589 968.426 RETICULOCALBIN 12 556.150 16INF NPNQNKNVAL 1111.586
1111.300 ELAV (HuR) 13 RPYSNVSNL SBF-1 14 LPQANRDTL MgcRacGap 15
QPRYPVNSV TCP-1 16 APAYSRAL HSP27 17 APKRPPSAF HMG-1 or HMG-2 18
AASKERSGVSL histone H1 19 family member Peptides Identified on
Uninfected cells that are not present on Infected cells 16UNINF
GSHSMRY MHC CLASS I 20 HEAVY CHAIN (could derive from multiple
alleles. I.e., HLA-B*0702 or HLA-G, etc.) SEQ START AA ACCESSION #
CATEGORY ID NO: Peptides Identified on Infected cells that are not
present on Uninfected Cells 101 HIV-DERIVED 1 250 2 152 10720340 3
MHC GENE PRODUCT 4 2 4566550 MHC GENE PRODUCT 5 UNKNOWN 6 527
4505939 RNA MACHINERY/BINDING PR 7 1073 Q04637 RNA
MACHINERY/BINDING PR 8 19 478522 TUMOR SUPPRESSOR GENE? 9 1823
13639246 TUMOR SUPPRESSOR GENE? 10 141 131528 RNA MACHINERY/BINDING
PR 11 4 4506457 TUMOR SUPPRESSOR GENE? 12 188 4503551 RNA
MACHINERY/BINDING PR 13 14 15 16 17 18 19 Peptides Identified on
Uninfected cells that are not present on Infected cells variable
multiple MHC Class I Product 20
was harvested for T cells infected and not infected with HIV.
Peptide ligands were eluted from B*0702 and comparatively mapped on
a mass spectrometer so that ions unique to infected cells were
apparent. Ions unique to infected cells (and one ligand unique to
uninfected cells) were subjected to mass spectrometric
fragmentation for peptide sequencing. Column 1 indicates the ion
selected for sequencing, column 2 is the HPLC fraction, column 3 is
the peptide sequence, column 4 is the predicted molecular weight,
column 5 is the molecular weight we found, column 6 is the source
protein for the epitope sequenced, column 7 is where the epitope
starts in the sequence of the source protein, column 8 is the
accession number, and column 9 is a descriptor which briefly
indicates what is known of that epitope and/or its source
protein.
[0080] The methodology used herein is to use sHLA to determine what
is unique to unhealthy cells as compared to healthy cells. Using
sHLA to survey the contents of a cell provides a look at what is
unique to unhealthy cells in terms of proteins that are processed
into peptides. The data summarized in TABLE I shows that the
epitope discovery technique described herein is capable of
identifying sHLA bound epitopes and their corresponding source
proteins which are unique to infected/unhealthy cells.
[0081] Likewise, and as is shown in Table I, peptide ligands
presented in individual class I MHC molecules in an uninfected cell
that are not presented by individual class I MHC molecules in an
uninfected cell can also be identified. The peptide "GSHSMRY" (SEQ
ID NO:20), for example, was identified by the method of the present
invention as being an individual class I MHC molecule which is
presented in an uninfected cell but not in an infected cell.
[0082] The utility of this data is at least threefold. First, the
data indicates what comes out of the cell with HLA. Such data can
be used to target CTL to unhealthy cells. Second, antibodies can be
targeted to specifically recognize HLA molecules carrying the
ligand described. Third, realization of the source protein can lead
to therapies and diagnostics which target the source protein. Thus,
an epitope unique to unhealthy cells also indicates that the source
protein is unique in the unhealthy cell.
[0083] The methods of epitope discovery and comparative ligand
mapping described herein are not limited to cells infected by a
microorganism such as HIV. Unhealthy cells analyzed by the epitope
discovery process described herein can arise from virus infection
or also cancerous transformation. In addition, the status of an
unhealthy cell can also be mimicked by transfecting a particular
gene known to be expressed during viral infection or tumor
formation. For example, particular genes of HIV can be expressed in
a cell line as described (Achour, A., et al., AIDS Res Hum
Retroviruses, 1994. 10(1): p. 19-25; and Chiba, M., et al., CTL.
Arch Virol, 1999. 144(8): p. 1469-85, all of which are expressly
incorporated herein by reference) and then the epitope discovery
process performed to identify how the expression of the transferred
gene modifies epitope presentation by sHLA. In a similar fashion,
genes known to be upregulated during cancer (Smith, E. S., et al.,
Nat Med, 2001. 7(8): p. 967-72, which is expressly incorporated
herein by reference) can be transferred in cells with sHLA and
epitope discovery then completed. Thus, epitope discovery with sHLA
as described herein can be completed on cells infected with intact
pathogens, cancerous cells or cell lines, or cells into which a
particular cancer, viral, or bacterial gene has been transferred.
In all these instances the sHLA described here will provide a means
for detecting what changes in terms of epitope presentation and the
source proteins for the epitopes.
Example of Epitope Binding Assay Using Radiolabeled Peptide and
Comparison of sHLA to Detergent Lysate-Prepared HLA
[0084] A specific assay for the A*0201T allele was used to test the
feasibility of a competitive binding assay to measure the binding
of defined synthetic antigenic peptides using sHLA class I
molecules. A peptide derived from HBV that is known to strongly
bind A*0201T was used to replace the endogenous peptide in
solution. An irrelevant p53 peptide was used as a negative control
in that it does not compete with the endogenous peptide for
specifically binding to A*0201T.
[0085] In the reaction, different concentrations of individual sHLA
having endogenous peptide bound therein was incubated with (1) a
standard concentration of HBV peptide (positive reaction), or (2)
p53 peptide (negative reaction), and after incubation for 48 hours
at room temperature, the sHLA complexes were immobilized on a solid
support using the HLA specific antibody W6/32. The A*0201T-HBV
peptide complex was detected using a highly specific antibody
(mouse anti-human MHC/HBV peptide antibody 5H9/1-2H) that only
recognizes this particular conformation (i.e., A*0201T with HBV
peptide bound therein), and A*0201T-endogenous peptide complexes
are not recognized by the antibody. To visualize the replacement
event, a secondary anti-mouse antibody conjugated to HRP was
used.
[0086] The assay was performed using an sHLA amount of 1.5 .mu.g
A*0201T at a final volume of 20 .mu.l (75 ng/.mu.l) and a final
peptide amount of 2 .mu.g at a final volume of 20 .mu.l (100
ng/.mu.l). After incubation, the reaction was 75.times. diluted,
and titrating amounts of sHLA-peptide complexes were captured with
the W6/32 antibody coated to an ELISA plate.
[0087] FIG. 3 illustrates that saturation of the W6/32 coated ELISA
plate could be achieved using the HBV peptide, demonstrating the
successful replacement of the endogenous peptide with the HBV
peptide. However, no saturation could be detected using the
irrelevant peptide p53, thereby supporting the specificity of HBV
peptide binding to sHLA A*0201T.
[0088] In addition, the sHLA molecules used herein were compared to
an HLA prepared by the prior art method of detergent lysate using
the above described method. FIG. 4 is a comparison of the
saturation curves of the sHLA allele A*0201T and the detergent
lysate preparation of A*0201 obtained through cell extraction. This
figure demonstrates that a much lower concentration of sHLA-A*0201
gives a detectable and useful signal as compared to the detergent
solubilized class I A*0201. This may be because the detergent
solubilized class I A*0201 is really a mixture of class I
molecules, or it may be because the detergent solubilized class I
does not have a full mixture of endogenous loaded peptides. The
peptides in the detergent solubilized A*0201 might represent only a
subset of the endogenously loaded peptides; purification of A*0201
from other class I in the lysate can bias the peptides.
[0089] However, the method of assaying epitope binding described
herein above is only applicable using the A*0201T allele loaded
with HBV because of the involvement of a highly specific antibody
recognizing only this specific conformation. Therefore, a more
direct measurement of epitope binding to sHLA has been identified
that eliminates the need to isolate and produce an antibody
specific for a particular peptide bound in each class I allele. The
method of Fluorescence Polarization involves labeling the peptide
of interest and will be described in detail herein below.
Epitope Binding Assay Using Fluorescence Polarization (FP)
[0090] One of the most attractive features of binding assays is
their simplicity. Developing a binding assay is to demonstrate that
the labeled probe binds to the molecule of interest. The following
series of criteria, however, must be met in order to validate the
binding assay: (1) binding should be saturable, indicating a finite
number of binding sites; (2) the binding should have the requisite
specificity, where the binding affinity, defined as the
dissociation constant (K.sub.d), should be consistent with values
determined for physiological molecules; and (3) ligand binding
should be reversible, reflecting the dynamic nature of the chemical
transmission process and reaching equilibrium when the ligand
association rate is equal to the dissociation rate.
[0091] However, while the execution of a routine binding assay
appears trivial, the development of such an assay requires many
hours of testing to establish validity. Numerous factors can
influence the specificity of ligand binding. Inorganic ions can
influence the attachment site of a ligand and factors such as time,
temperature, pH, and peptide concentration influence the kinetic
properties, and possibly the specificity, of a binding assay. Other
considerations include the stability of the fluorescent dye in the
incubation medium and, even more fundamentally, the biological
activity of the peptide.
[0092] Fluorescein is one of the most often used molecules to
produce fluorescent labels to localize antigen, receptors or other
moieties with affinity for the labeled molecules. FITC (fluorescein
isothiocyanate) is very fluorescent in aqueous environments and it
gives high sensitivity under UV light; however, its fluorescence is
pH-dependent. Its green fluorescence disappears in acid, but
reappears in neutral or basic solutions. The pK.sub.a of FITC is
approximately 6.5, and it gives its best fluorescent signal above
pH 8. FITC has a molecular weight (MW) of 473.4; excitation and
emission wavelengths at 494 nm and 520 nm, respectively; and a
molar extinction coefficient of 72,000 M.sup.-1 cm.sup.-1 in an
aqueous buffer, pH 8. Thus, FITC shifts in color from a
blue-to-green fluorescent product. FITC fluorescence has a tendency
to fade, but this fading may be overcome through the use of agents
such as n-propyl gallate or phenylenediamine to stabilize the
color. However, these reagents are toxic to living cells and cannot
be used in all applications. Isothiocyanates also react with a
variety of metal ions to form colored complexes. Therefore, metals
such as Fe.sup.3+, Cu.sup.1+, and Ag.sup.1+ should be avoided in
reaction mixtures.
[0093] Fluorescence is characterized by a process of absorption of
incident radiation at a wavelength, .lamda..sub.abs, followed by
the emission of radiation at another wavelength, .lamda..sub.emiss.
This behavior was first described by G. G. Stokes (1852) in the
form of Stokes' Law of Fluorescence in which he stated that
fluorescence (emission) always appears at a wavelength greater than
the wavelength of the incident (excitation) radiation.
[0094] This behavior was successfully explained by A. Einstein
(1905) using Planck's quantum hypothesis. A quantum of incident
light with an energy of E.sub.excit is absorbed by the fluorescent
molecule raising its energy to E.sub.excit. This is quickly
followed by a downward transition of the molecule to one of the
vibration levels in the ground state, E.sub.emiss, with the
emission of a quantum of light.
[0095] The phenomenon of absorption and fluorescence can be
described in terms of an energy level diagram showing the
transitions between the vibration levels of the ground state
(s.sub.0) and the vibration levels of the first excited state
(s.sub.1).
[0096] In order to describe fluorescence and fluorescence
polarization an incident beam strikes a fluorescent molecule
(absorption) whereupon the molecule then emits fluorescent
radiation. The parallel and vertical intensities of this
fluorescence are represented by I.sub..parallel. and I.sub.,
respectively, which serve as the components used in the
experimental configuration to observe fluorescence
polarization.
[0097] The primary parameter used in the field of the fluorescence
polarization to describe the polarization of fluorescence is the
degree of polarization, P. This parameter is a very useful way of
summarizing the polarization state of fluorescence. The equation
for the degree of polarization, P is:
Polarization=(I.sub..parallel.-I.sub.)/(I.sub..parallel.+I.sub.)
where I.sub. is the intensity of the fluorescence measured in the
perpendicular () or vertical (V) direction and I.sub..parallel. is
the intensity of the fluorescence measured in the parallel
(.parallel.) or horizontal (H) direction. These measurements are
carried out using a polarizer rotated to the vertical and
horizontal directions, respectively.
[0098] By using a fluorescent dye to label a small molecule, its
binding to another molecule of equal or greater size can be
monitored using fluorescence polarization (FP). FP operates on the
principle that small molecules rotate faster than large molecules.
If a molecule is labeled with a fluorophore, this rotation can be
measured by exciting the fluorophore with plane polarized light and
then measuring the emitted light with polarizers parallel and
perpendicular to the plane of excitation to determine if it is
still oriented in the same plane as it was when excited. If a
fluorophore is labeled on a small molecule it will rotate in the
time between excitation and emission and the light emitted will be
depolarized. If the labeled molecule binds to a large molecule
(effectively increasing its overall size) the molecule will not
rotate in the time between excitation and emission, and the light
emitted will be polarized resulting in a polarization change
between the free and bound forms. For convenience, units are
usually 1000 mP=P.
[0099] The maximum theoretical mP value obtainable is 500 mP. Hence
any experimental value greater than this suggests an artifact
within the assay. The assumed theoretical mP for fluorescein is 27.
When a small free tracer is bound to a large molecule, the mP is
expected to increase. A good FP assay usually has an mP change of
100 or more.
[0100] A number of factors can contribute to the lowering of the
maximum obtainable polarization value. Some factors that can
influence this are quenching of the fluorophore by the molecules
themselves, buffer quenching, adsorption onto surfaces, rotational
spin (the "propeller effect") and low affinity of interaction
between the components.
[0101] Polarization methods are used to measure affinities of
FITC-labeled peptide probes for purified sHLA molecules.
Equilibrium results contributed to the prediction of a efficacious
dose (IC.sub.50) and clarified the strong correlation of in vitro
potency to the form of the sHLA molecule used in the assay. Such
results also demonstrated that equilibrium polarization
measurements are feasible after optimization of assay parameters.
In addition, kinetic measurements are possible with FP.
[0102] Advantages of FP measurements include: homogeneous
measurements, equilibrium and kinetic binding data, plate readers
available with improved sensitivity and reduced minimum volume for
detection, and competitor affinity data. FP uses one label only and
has a truly homogeneous format, i.e., no solid phase to prepare or
suspend and no washing steps required, and rapid kinetic data can
be obtained. In addition, no radioactivity is required, and the
ratiometric reading resists color quenching.
[0103] Minimization of the contribution of assay components to
non-specific fluorescence polarization is very important. Quality
factors include purity of tracer (fluorescent peptide), purity of
binder (sHLA), buffer intrinsic fluorescence and ability of buffer
components such as carrier proteins to bind the tracer. Some
microplate materials such as polystyrene can bind free tracer,
thereby increasing the polarization.
[0104] Because of the ease with which fluorescence can be detected
it is also important to know that it is the compound of interest
that is being traced and not a fluorescent impurity. Thus,
manufacturers provide data on the specificity and purity of their
products. Tracer should be >90% labeled and >95% pure.
Failure to purify free fluorophore from tracer means an increased
portion of the total fluorescence will not be able to change its
polarization.
[0105] The dye most commonly used for labeling peptides is
fluorescein. A fluorescent labeled substance is biologically not
identical to the unlabeled compound and it may not behave in a
manner similar to the parent molecule.
[0106] Because large proteins, cell membranes and cellular debris
scatter light, causing a net increase in total polarization,
impurities should be minimized, and only highly purified binder
should be used. In some cases, the impurities may be corrected in
part by appropriate background subtraction, but it is preferable to
minimize the contribution to signal by using purified
molecules.
[0107] A mixture of protease inhibitors has to be considered if
degradation occurs during incubation. A simple cocktail is often
sufficient to protect the molecule, such as the use of a protease
inhibitor cocktail (Gibco BRL Cat# 20012-043) dissolved in PBS pH
7.4 or a final concentration of 1 mM PMSF, 73 mM pepstatin A, and 8
mM EDTA. In the sHLA assay, highly purified sHLA molecules will be
used which are also not susceptible to degradation through
proteases and thus do not require the addition of protease
inhibitors or calcium-chelating salts (EDTA or EGTA) to the buffer
system.
[0108] In addition, buffer contribution to signal should be
minimized. Increased buffer fluorescence background is due to
contaminants that fluoresce at the wavelength of interest.
Attention to raw materials, cleanliness of mixing and storage
vessels and buffer preparation methods should reduce this to
acceptable levels. High background counts due to buffer or
non-fluorophore components can seriously affect the signal-to-noise
ratios of an assay as well as the ultimate sensitivity of an
assay.
[0109] Buffers for proteins often include carrier proteins, such as
bovine albumin (BSA). Albumin may bind some fluorophores, and
binding of BSA to the tracer could spuriously increase the baseline
polarization, thereby reducing assay range. However, this problem
can be overcome by avoiding carrier proteins or using low binding
alternatives such as bovine gamma globulin (BGG).
[0110] In any case, it is useful to evaluate the contribution of
buffer proteins to the net polarization of the tracer by comparing
polarization in buffer with and without added protein.
Alternatively, the final concentration of BSA can be reduced to
minimize these effects.
[0111] Finally, likely sources of imprecision which increase the
standard deviation of the assay include pipeting, instrument,
buffer, tracer, and protein; each component contributes to total
imprecision.
[0112] A critical feature of the recombinant sHLA molecule is the
ability to load the peptide binding portion with a peptide of
interest. Results obtained from acid eluted peptide pools indicate
that the majority of recombinant sHLA complexes are folded around
undefined "bulk peptides" derived from the cell line in which they
are produced. It is necessary to replace these endogenous peptides
with single, well-defined, labeled standard peptides. Endogenous
sHLA molecules having endogenous peptide or no peptide bound will
probably be present in a majority, but will not have any
interference with the outcome of the assay. The peptide should be
highly purified, as small contaminants in synthesized peptides can
inhibit peptide loading.
[0113] Several peptide loading protocols have been described. A
simple method involves passive loading of excess peptide in
solution with sHLA. Passive loading works particularly well in the
case of high-affinity peptides. For lower-affinity peptides, an
increase in the molar ratio of peptide to HLA may improve loading.
For each peptide, parameters such as the dose of HLA, molar ratio
of peptide to HLA and peptide loading time need to be empirically
determined by the investigator.
[0114] In order for the MHC class I alleles to be capable of
binding peptides, recent peptide loading experiments indicated that
.beta.2-microglobulin (.beta.-2-m) must be present. In general, MHC
class I molecules are passively loaded over a several-day time
course (in a range of from about 2 to about 5 days). Optimal
peptide loading may vary for specific MHC class I alleles. While
passively loaded complexes are generally sufficient to work with,
they are not necessarily optimally loaded. Parameters and minimal
requirements for peptide binding to HLA have been reported. (Khilko
S N, et al. Measuring interactions of MHC class I molecules using
surface plasmon resonance. J Immunol Methods 183(1):77-94 (1995);
Parker K C, et al. Peptide binding to HLA-A2 and HLA-B27 isolated
from Escherichia coli. Reconstitution of HLA-A2 and HLA-B27 heavy
chain/beta 2-microglobulin complexes requires specific peptides. J
Biol Chem. 267(8):5451-9 (1992); Parker K C, et al. Sequence motifs
important for peptide binding to the human MHC class I molecule,
HLA-A2. I Immunol. 149(11):3580-7 (1992), all of which are hereby
expressly incorporated herein by reference).
[0115] HLA complexes are also successfully loaded by a short
alkaline stripping procedure followed by slow refolding at neutral
pH. Peptide stripping can also be done in the presence of excess
.beta.-2-m under mildly acidic conditions.
[0116] There are three basic types of binding experiments: (1)
saturation experiments in which a saturation curve is generated, by
holding either the amount of fluorescent peptide (tracer) or sHLA
(binder) constant and varying the concentration of sHLA in case of
constant tracer or the labeled peptide in case of constant binder
in order to determine the affinity constant (K.sub.d); (2)
competition experiments in which the amount of a competing,
unlabeled compound for the receptor, included in the incubation, is
the only variable, and the affinity (K.sub.i) of that drug can be
determined; and (3) kinetic experiments from which the forward
(k.sub.on) and reverse (k.sub.off) rate constants of the binding
process can be determined if the amount of sHLA and peptide are
held constant and the time varied. The ratio of these constants
provide an independent estimate of the K.sub.d.
Description of Saturation Experiments
[0117] Passive loading of excess peptide in solution with sHLA is
used in the saturation assays. For each peptide, parameters such as
the dose of HLA, molar ratio of peptide to HLA and peptide loading
time need to be empirically determined. MHC class I molecules are
passively loaded over a several-day time course (in a range of from
about 2 to about 5 days). Optimal peptide loading may vary for
specific MHC class I alleles.
Titration Assay to Establish Optimal sHLA Concentration
[0118] The assay will determine the sHLA concentration necessary to
yield a sufficient peptide binding. Specific binding of various
concentrations of sHLA (dose range: 0.1 nM-1000 nM) in the presence
of a fixed concentration (5 nM) of fluorescent-labeled synthetic
peptide should be tested. The fixed fluorescent-labeled synthetic
peptide should be evaluated in preliminary experiments including
biochemical considerations: the concentration of the tracer should
be less than the K.sub.d and less than the concentration of
available binder (SHLA) allowing peptide binding. It is recommended
that the binder (sHLA) should be at a higher concentration than
tracer (fluorescent-labeled synthetic peptide).
[0119] Comparison of free tracer with free fluorophore (by running
free fluorescein in parallel) establishes the suitability of tracer
size. If the tracer mP is significantly greater than that of the
comparable fluorophore, the tracer may not be optimal for the use
in FP. For adequate net polarization change, evaluation of several
tracers must be conducted.
[0120] The purpose of titrating binder (sHLA) with appropriate
controls is to determine the optimal concentration of binder and
tracer. The acceptable range of concentrations of tracer include
all concentrations giving a polarization value (in mP) near to the
prescribed 27 mP. If the integration time is 100,000 microseconds,
the counts per second should be at least 100,000.
[0121] Use of appropriate controls allows accurate estimation of
specific polarization. The background signal is the contribution to
the measurement from sources other than the fluorescent label. It
is most easily seen taking a measurement on a well containing all
test components except the fluorescent label. Background signals
may arise from the microplate plastic, solution contaminants,
leakage of light through the optical filters, or other sources
generated in the instrument. If the background is highly
predictable, i.e., constant from well to well, it can largely be
eliminated by subtracting the signal from a control well lacking
fluorescent label. Subtracting this constant signal will yield
useful information from wells generating signals that are close to
background levels.
[0122] Specific control groups utilized in the assay include: (1)
Buffer only, (2) Tracer only, (3) Protein only and (4)
Protein+Tracer. The purpose of each of the specific control groups
is discussed in detail herein below.
[0123] The "Buffer only" control indicates the contribution of
buffer alone to the S and P signals, especially when interfering
molecules are present in the buffer. Since the binder may
contribute to net signal, binder without tracer serves as a proper
control and is used as background subtraction for "Tracer only".
For multiple concentrations of binder, each should have the "Buffer
only" control.
[0124] In the "Tracer only" control, S and P values for free tracer
are background-subtracted with "Buffer only" controls and used for
G factor calculation. G factors are calculated using the assumed
theoretical mP (27 for fluorescein). S (parallel) and P
(perpendicular) are the background subtracted intensity
measurements when the polarizers are in parallel or perpendicular
direction. G .times. .times. factor = S P * ( 1 - 27 1000 ) ( 1 +
27 1000 ) ##EQU1##
[0125] G should be a very stable value which corrects for the
contribution of the measurement pathway to the observed total
polarization. Optical pathways, particularly those with reflective
components, pass light of different polarization with varying
efficiencies. Instrumental elements that impact this correction
factor include the dichroic mirror, excitation and emission
filters, polarizing filters and attenuators. Other elements that
influence the G factor include the assay plate and buffer/assay
components.
[0126] Essentially the G factor functions as a scaling (correction)
factor, taking relative polarization measurements and making them
appear absolute (relative to a known standard). The G factor is
typically a value ranging from 0.8 to 1.2. Obtaining a G factor
very different from 1 suggests that the filters and dichroic mirror
may not be optimized for fluorescence polarization, although
meaningful results may be obtained.
[0127] Once a G factor has been determined, it can be entered into
the associated fluorescence polarization method. The calculated mP
from the Criterion Host software will now report "corrected" mP
values. Using the established G factor, calculate the mP value for
the free tracer. The mP values are generally obtained by
subtracting the mean S and P background values from the individual
S and P values of the free tracer. As a control, the free
fluorophore should have an mP value close to the theoretical value
and serves as the minimal polarization value (mP.sub.min). mP = S -
( P * G ) S + ( P * G ) * 1000 ##EQU2##
[0128] The "Protein only" control indicates contribution of light
scattering by the specific protein binder. This is used as
background subtraction for "Protein+Tracer". Since several
concentrations of protein will be used, each should be tested in
the absence of tracer.
[0129] For "Protein+Tracer", the S and P values are background
subtracted with "Protein only" controls to determine the maximal mP
for a given concentration. For background subtraction, the mean S
and P "Protein only" values are calculated, and the appropriate
mean from individual S and P values of wells containing
"Protein+Tracer" are subtracted. The calculated G factor is used.
mP = S - ( P * G ) S + ( P * G ) * 1000 ##EQU3##
[0130] As an additional check on the system, it is advisable to
re-read the plate in the Fluorescence Intensity mode. If the same
amount of tracer is present in each well, then there should be
equal intensity values across the plate in the Fluorescence
Intensity mode. Re-reading the plate in intensity mode allows
evaluation of the extent of quenching. Quenching effects can affect
the ultimate sensitivity of a fluorescence-based assay. The
comparison of the molar fluorescence intensity of the
fluorophore-labeled peptide and the fluorophore itself in free
solution can be used to determine the degree of quenching caused by
the chemical coupling process itself. If there is no quenching, the
signal for 1 nM fluoresceinated peptide should be the same as that
of the free fluorescein. It is not expected that fluorescein
coupled to another molecule would be more fluorescent than free
fluorescein, so this could indicate that the tracer may have an
incorrect concentration assigned. Fluorescence polarization often
results in the loss of about 10-90% of fluorescence intensity. This
in itself may reduce the sensitivity of fluorescence polarization
as opposed to direct intensity measurements.
Method of Epitope Binding Assay Using Flouorescence
Polarization
[0131] The FITC-labeled peptides are prepared by dissolving the
lyophilized powder in 90% Acetonitrile/10% water to a final
concentration of 0.25 mM (this measurement should be as precise as
possible). It is important to make sure everything is gone into
solution. If there is still precipitate present, incubate overnight
at room temperature with shaking. Seal the tube with parafilm and
protect from light. (Some peptides will dissolve only by adding
NH.sub.4HCO.sub.3 at a final concentration of 25 mM to convert the
acidic environment into a more basic state).
[0132] The FITC labeled peptide solution is divided into 800 .mu.l
aliquots and added to sealable screw cap tubes. The aliquots are
then lyophilized overnight at room temperature. Each tube should
finally contain 0.2 .mu.mol of peptide. Each tube is closed
tightly, and parafilm is used to wrap the top. The tube is then
labeled appropriately and stored at -80.degree. C. until use.
[0133] For an adequate net polarization change, several
FITC-labeled peptide tracers are evaluated. For optimal evaluation
of FP data, 5 independent reaction types need to be mixed, as
described in Table II. TABLE-US-00002 TABLE II A Reaction Mix
"Protein + peptide sHLA; .beta.-2-m; pFITC; Tracer" PBS; (BGG) B
Protein only control "Protein only" sHLA; .beta.-2-m; PBS; (BGG) C
Labeled peptide only "Free Peptide Tracer pFITC; .beta.-2-m; PBS;
control only" (BGG) D Free FITC tracer only "Free FITC Tracer FITC;
.beta.-2-m; PBS; control only" (BGG) E Buffer only control "Buffer
only" .beta.-2-m; PBS; (BGG)
[0134] Multiple concentrations of sHLA are tested for specific
binding in a range of 0.1-1000 nM. A first study may include rather
broad concentration ranges for both tracer and binder, whereas a
follow-up test may use only one concentration of tracer and a
tightly spaced limited dilution series of the binder.
TABLE-US-00003 HC (A*0201T) 273 aa (31,495) N-glycosylation --
(3,000) .beta.-2-m 99 aa (11,731) Peptide (9mer) 9 aa (1,000)
Complex 381 aa (47,226) 47.2 .mu.g/ml 1 .mu.M 47.2 ng/ml 1 nM
[0135] In order for the MHC class I alleles to be capable of
binding peptides, recent peptide loading experiments indicated that
additional .beta.2-microglobulin must be present. The addition of
an extra amount of .beta.-2-m in a ratio of .beta.-2-m:sHLA between
about 0.5 to about 2.0 are reasonable. Desirably, a ratio of
.beta.-2-m:sHLA of about 1.0 is utilized in the beginning and then
adjusted if required. TABLE-US-00004 .beta.-2-m 99 aa (11,731) 11.7
.mu.g/ml 1 .mu.M 11.7 ng/ml 1 nM
[0136] Suggested dilutions of sHLA and .beta.-2-m are as shown in
Table III: TABLE-US-00005 TABLE III sHLA dilutions .beta.-2-m
dilutions 1 50,000 ng/ml 1060 nM 12,402 ng/ml 1060 nM 2 10,000
ng/ml 212 nM 2,480 ng/ml 212 nM 3 5,000 ng/ml 106 nM 1,240 ng/ml
106 nM 4 1,000 ng/ml 21.2 nM 248 ng/ml 21.2 nM 5 500 ng/ml 10.6 nM
124 ng/ml 10.6 nM 6 250 ng/ml 5.3 nM 62 ng/ml 5.3 nM 7 125 ng/ml
2.65 nM 31 ng/ml 2.65 nM 8 62.5 ng/ml 1.32 nM 15.5 ng/ml 1.32 nM 9
31.25 ng/ml 0.66 nM 7.75 ng/ml 0.66 nM 10 15.6 ng/ml 0.33 nM 3.88
ng/ml 0.33 nM 11 7.8 ng/ml 0.16 nM 1.94 ng/ml 0.16 nM 12 3.9 ng/ml
0.08 nM 0.97 ng/ml 0.08 nM
[0137] To prepare 2.times. dilutions of sHLA+excess .beta.-2-m, a
total volume of 550 .mu.l is sufficient to perform 5 independent
measurements for A ("Protein+Peptide tracer") and B ("Protein
only") with a backup volume of 50 .mu.l. The used dilution scheme
will use up 81.4 .mu.g of sHLA and 20.2 .mu.g .beta.-2-m for the
(A) & (B) reactions.
[0138] PBS pH 7.4 is used as the buffer. Optionally, 0.05% (0.5
mg/ml) bovine gamma a globulin (BGG) may be used as supplement to
prevent non-specific binding of protein on tube walls or as carrier
protein. When using BGG, prepare a volume of 33 ml of freshly mixed
PBS/0.05% BGG (16.5 mg BGG/33 ml PBS).
[0139] To prepare the 2.times. dilutions, twelve "non-stick" 1.5 ml
tubes are labeled with the numbers 1-12. Table IV below describes
the dilutions. Each tube is mixed thoroughly after adding
sHLA/.beta.-2-m. Careful pipetting is required.
[0140] The first tube (tube #1) should originally contain 814 .mu.l
total volume with a sHLA concentration of 100,000 ng/ml and a
.beta.-2-m concentration of 24,804 ng/ml. Therefore, the amounts of
sHLA and .beta.-2-m to be added to tube #1 are calculated, and then
the tube is filled up to 814 .mu.l. .mu. .times. .times. l .times.
.times. sHLA .times. .times. to .times. .times. add = 100 .times.
.times. .mu. .times. .times. g .times. / .times. ml .times. .times.
final .times. .times. conc . * 814 .times. .times. .mu. .times.
.times. l .times. .times. total .times. .times. volume [ mg .times.
/ .times. m .times. .times. l .times. .times. stock .times. .times.
sHLA ] .mu. .times. .times. l .times. .times. b2m .times. .times.
to .times. .times. add = 24.8 .times. .times. .mu. .times. .times.
g .times. / .times. ml .times. .times. final .times. .times. conc .
* 814 .times. .times. .mu. .times. .times. l .times. .times. total
.times. .times. volume [ mg .times. / .times. m .times. .times. l
.times. .times. stock .times. .times. b2m ] ##EQU4## TABLE-US-00006
TABLE IV Volume of Volume of Tube # or sHLA/.beta.-2-m Tube
PBS/0.05% BGG stock sample 2.times. dilutions # to add to use to
add (ng/ml) 1 see above see above see above 100,000/24,804 2 1056
.mu.l Tube 1 264 .mu.l 20,000/4,961 3 770 .mu.l Tube 2 770 .mu.l
10,000/2,480 4 880 .mu.l Tube 3 220 .mu.l 2,000/496 5 550 .mu.l
Tube 4 550 .mu.l 1,000/248 6 550 .mu.l Tube 5 550 .mu.l 500/124 7
550 .mu.l Tube 6 550 .mu.l 250/62 8 550 .mu.l Tube 7 550 .mu.l
125/31 9 550 .mu.l Tube 8 550 .mu.l 62.5/15.5 10 550 .mu.l Tube 9
500 .mu.l 31.25/7.75 11 550 .mu.l Tube 10 550 .mu.l 15.6/3.88 12
550 .mu.l Tube 11 550 .mu.l 7.8/1.94
[0141] To prepare 2.times. control dilutions of excess .beta.-2-m
only, a total volume of 800 .mu.l is sufficient to make up the rest
of the controls necessary for C ("Free peptide tracer only"), D
("Free FITC tracer only") and E ("Buffer only") with a backup
volume of 50 .mu.l. The used dilution scheme will use about 29.4
.mu.g .beta.-2-m for the (C), (D) & (E) reaction.
[0142] To prepare the 2.times. dilutions, twelve "non-stick" 1.5 ml
tubes are labeled with the numbers 1-12. Table V describes the
dilutions. Each tube is mixed thoroughly after adding
sHLA/.beta.-2-m. Careful pipetting is required.
[0143] The first tube (tube #1) should originally contain 1184
.mu.l total volume with a .beta.-2-m concentration of 24,804 ng/ml.
The amount of .beta.-2-m to be added to tube #1 is calculated, and
then the tube is filled to 1184 .mu.l. .mu. .times. .times. l
.times. .times. b2m .times. .times. to .times. .times. add = 24.8
.times. .times. .mu. .times. .times. g .times. / .times. ml .times.
.times. final .times. .times. conc . * 1184 .times. .times. .mu.
.times. .times. l .times. .times. total .times. .times. volume [ mg
.times. / .times. m .times. .times. l .times. .times. stock .times.
.times. b2m ] ##EQU5## TABLE-US-00007 TABLE V Volume of PBS/0.05%
Volume of Tube BGG Tube # or stock .beta.-2-m sample 2.times.
dilutions # to add to use to add (ng/ml) 1 see above see above see
above 24,804 2 1536 .mu.l Tube 1 384 .mu.l 4,961 3 1120 .mu.l Tube
2 1120 .mu.l 2,480 4 1280 .mu.l Tube 3 320 .mu.l 496 5 800 .mu.l
Tube 4 800 .mu.l 248 6 800 .mu.l Tube 5 800 .mu.l 124 7 800 .mu.l
Tube 6 800 .mu.l 62 8 800 .mu.l Tube 7 800 .mu.l 31 9 800 .mu.l
Tube 8 800 .mu.l 15.5 10 800 .mu.l Tube 9 800 .mu.l 7.75 11 800
.mu.l Tube 10 800 .mu.l 3.88 12 800 .mu.l Tube 11 800 .mu.l
1.94
[0144] Finally, a stock solution of FITC-labeled peptide tracer
(pFITC) is prepared using a pre-measured tube (0.2 .mu.mol) and
stored at -80.degree. C. 20 .mu.l of DMSO is added to k0.2 .mu.mol
of FITC-labeled peptide powder pre-measured in a microtube to
receive a stock concentration of 10 mM and pipetted up and down
until the tracer is completely dissolved. Optionally, 20 .mu.l of
DMF may be used for peptides containing methionine (M) which are
known to oxidize more likely in DMSO than DMF. To protect from
moisture, the FITC-labeled peptide and DMSO are allowed to
equilibrate to room temperature before opening.
[0145] To obtain a 100 .mu.M working dilution, 980 .mu.l of PBS is
added to the original DMSO stock, and then the working
concentration is further diluted to a 1 .mu.M solution (10 .mu.l
working dilution to 990 .mu.l PBS). The 100 .mu.M working dilution
is aliquoted and stored at -25.degree. C.
[0146] Acceptable concentrations of FITC-labeled peptides in FP
experiments are in a 0.75 to 50 nM range. A mP range of free FITC
tracer is expected to be between 30-50 mP. Intensities for parallel
and perpendicular fluorescence for the concentration range of 0.75
to 50 nM FITC is 0.3 to 30 10.sup.6 cps. Unpolarized fluorescence
intensity will be 10.times. over the polarized intensities. An
optimal starting concentration of FITC-labeled peptide would be 5
nM. Consider running more than one concentration of tracer
initially.
[0147] To prepare 20 ml of 5 nM pFITC used in (A) ("Protein+Peptide
tracer") and (C) ("Free peptide tracer only"), 100 .mu.l of the 1
.mu.M pFITC is added to 19.9 ml buffer. Free peptide tracer is
compared with free fluorophore by running free FITC in parallel to
establish the suitability of the tracer and as a control if the
correct concentration was assigned to the peptide tracer. If there
is no quenching the signal for the selected fluoresceinated peptide
concentration should be the same as that of the FITC control.
[0148] A stock solution of FITC is prepared using pre-measured FITC
(Fluorescein Isothiocyanate) Microtubes (Pierce # 51004; 6.times.1
mg). (1 mg/ml is 2.112 mM). FITC has a molecular weight (MW) of
473.4. 211.2 .mu.l of DMSO is added to 1 mg of FITC powder
pre-measured in a microtube to receive a stock concentration of 10
mM. The FITC labeling reagent is reconstituted by puncturing the
foil and adding 211.2 .mu.l of DMSO and pipetting up and down until
the FITC is completely dissolved. To protect from moisture, the
FITC and DMSO are allowed to equilibrate to room temperature before
opening.
[0149] To obtain a 100 .mu.M working dilution, 980 .mu.l of PBS is
added to the original DMSO stock. The working concentration is
further diluted to a 1 .mu.M solution (10 .mu.l working dilution to
990 .mu.l PBS). The 100 .mu.M working dilution is aliquoted and
stored at -25.degree. C.
[0150] To prepare 10 ml of 5 nM pFITC used in (D) ("Free FITC
tracer only"), 50 .mu.l of the 1 .mu.M pFITC is added to 9.95 ml
buffer.
[0151] To start the peptide exchange reaction, mix 50 .mu.l of each
solution prepared above according to the scheme shown in FIG. 5. 50
.mu.l buffer (PBS/(BGG) is added in row 1-12 of B & E. 50 .mu.l
of FITC solution is added in row 1-12 of D, and 50 .mu.l of pFITC
is added in row 1-12 of A & C. The serial dilutions of the
.beta.-2-m series are added only to C, D & E starting with 12
and ending with 1. Finally, the serial dilutions of the
sHLA/.beta.-2-m series are added to A & B, also starting with
12 and ending with 1. The dilutions are then kept at 4.degree. C.
in the dark.
[0152] The meniscus are checked to make sure they are uniform and
are evened out by gentle tapping if necessary. Air bubbles, which
may be present in some wells, are removed.
[0153] The usual incubation time for a peptide exchange reaction is
about 48 hours. However, to determine optimal binding times, the
course of binding should be observed, by reading the plate several
times during incubation. Start the first reading at t=0 hours.
[0154] A fluorescence polarization detection method is prepared
with the following parameters:
[0155] Lamp: continuous lamp
[0156] Plate format: as appropriate for the plate to be
evaluated
[0157] Switch polarization after each well
[0158] Select wells: specify all the wells to be read
[0159] G factor: 1
[0160] Z Height: 1 mm
[0161] Filters: fluorescein excitation and emission (also use the
fluorescein dichroic in the top optics head)
[0162] Timing, Continuous Lamp: [0163] Readings per Well: 1 [0164]
Integration Time: 100,000 .mu.sec
[0165] Raw Data Units: counts/sec
[0166] Attenuator mode: out
[0167] Polarizers: excitation polarizer in the S position emission
polarizer as dynamic
[0168] PMT setup: SmartRead, sensitivity 2
[0169] Plate agitation: none
[0170] Kinetic timing: [0171] Delay Before First Read: 0 [0172]
Delay Between Reads: 0 [0173] Number of Reads: 1.
[0174] Plate Settling Time: 25 ms
[0175] The plate is re-read in Fluorescence Intensity mode to
determine the degree of quenching caused by the chemical coupling
process of sHLA and peptide. Without quenching and equal amount of
tracer present in each well, the intensity values across the plate
should be the same. Note that fluorescence intensity reaches a
10-90% higher signal than fluorescence polarization.
[0176] A fluorescence intensity detection method is then prepared
with the following parameters:
[0177] Optics: top, continuous lamp
[0178] Plate format: as appropriate for the plate to be
evaluated
[0179] Select wells: specify all the wells to be read
[0180] Z Height: 1 mm
[0181] Filters: fluorescein excitation and emission (also use the
fluorescein dichroic in the top optics head)
[0182] Timing, Continuous Lamp: [0183] Readings per Well: 1 [0184]
Integration Time: 100,000 .mu.sec
[0185] Raw Data Units: counts/sec
[0186] Attenuator mode: out
[0187] Polarizers: none
[0188] PMT setup: SmartRead, sensitivity 2
[0189] Plate agitation: none
[0190] Kinetic timing: [0191] Delay Before First Read: 0 [0192]
Delay Between Reads: 0 [0193] Number of Reads: 1.
[0194] Plate Settling Time: 25 ms
[0195] After reading the plate, the plate is covered with a lid and
sealed with parafilm. To protect from light, the plate is
additionally covered with aluminum foil. The plate is then
incubated at assigned temperature until next reading. Each reading
is recorded as follows: TABLE-US-00008 Date: Time: No. Incubation
period Current incubation time t.sub.1 0 hours 0 hours t.sub.2
hours hours t.sub.3 hours hours t.sub.4 hours hours t.sub.5 hours
hours t.sub.6 hours hours t.sub.7 hours hours t.sub.8 hours hours
t.sub.9 hours hours t.sub.10 hours hours
[0196] The data is evaluated using a spread-sheet program, such as
Microsoft's EXCEL.TM.. The net increase in polarization is
determined upon addition of sHLA to the peptide. The "Free peptide
tracer only" values provide the basis to calculate the G factor.
"Tracer only" gives the lowest attainable mP (minimum tracer
binding) not using a G factor to correct values to a theoretically
assumed value. The "Buffer only" control provides a background
control, indicating the contribution of buffer only to the S and P
signals, especially when interfering molecules are present in the
buffer. The background signal is the contribution to the
measurement from sources other than the fluorescent label. It is
most easily seen taking a measurement on a well containing all test
components except the fluorescent label. Background signals may
arise from the microplate plastic, solution contaminants, leakage
of light through the optical filters, or other sources generated in
the instrument. S and P values for "Free peptide tracer only" and
"Free FITC tracer only" are background-subtracted with "Buffer
only" controls before calculating mP and G factor.
[0197] If there are several concentrations of (unlabeled)
components present (i.e., multiple .beta.-2-m concentrations),
there should be a background well for each concentration.
Experiments with homogenous buffer systems are background corrected
by subtracting the mean values.
[0198] G factors are calculated using the assumed theoretical mP
(27 for fluorescein). S (parallel) and P (perpendicular) are the
background subtracted intensity measurements when the polarizers
are in parallel or perpendicular direction. G .times. .times.
factor = S P * ( 1 - 27 1000 ) ( 1 + 27 1000 ) ##EQU6##
[0199] Using the calculated G factor will adjust all results to the
assumed theoretical mP of 27 as the lowest attainable mP value
(minimum tracer binding). The signal to background ratios are
determined to assure the quality of the G factor. [0200] "Free
peptide tracer only"/"Buffer only" [0201] and [0202] "Free FITC
tracer only"/"Buffer only" A representative value for
signal/background can be calculated from the Parallel (S) value of
signal "Free peptide tracer only" to background "Buffer only"
values. Ideally, signal to background values of at least 10-fold
should be targeted. Errors become usually large at low
concentration as background noise dominates the intensities.
[0203] Noise relates to the uncertainty in measurements and usually
determines the ultimate sensitivity of an instrument. To determine
signal to noise ratios, the mP values are divided by the standard
deviation where the mP value corresponds to signal and the
imprecision (standard deviation) corresponds to the "noise".
Ideally, signal to noise values of at least three times the
background standard deviation should be targeted.
[0204] As additional check on the system, it is advisable to
re-read the plate in the Fluorescence Intensity mode. Re-reading
the plate in intensity mode allows evaluation of the extent of
quenching. Quenching effects can affect the ultimate sensitivity of
a fluorescence-based assay.
[0205] The comparison of the molar fluorescence intensity of the
fluorophore-labeled peptide and the fluorophore itself in free
solution can be used to determine the degree of quenching caused by
the chemical coupling process itself. Compare "Free peptide tracer
only" fluorescence intensities with "Free FITC tracer only"
fluorescence intensities re-read in fluorescence intensity mode. If
there is no quenching, the signal for 1 nM fluoresceinated peptide
should be the same as that of the free fluorescein. It is not
expected that fluorescein coupled to another molecule to be more
fluorescent than free fluorescein, so this could indicate that the
tracer may have an incorrect concentration assigned. In addition,
the obtained fluorescence intensity results are confirmed by G
factor comparison between "Free peptide tracer only" and "Free FITC
tracer only".
[0206] The S and P "Protein+Peptide tracer" values determine the
highest attainable mP (maximum tracer binding) fora chosen
concentration. Before calculating mP, the background is subtracted
with "Protein only" controls, and the calculated G factor is
used.
[0207] The "Protein only" control indicates the contribution of
light scattering by the specific protein binder. Since several
concentrations of protein will be used, each should be tested in
the absence of tracer. mP = S - ( P * G ) S + ( P * G ) * 1000
##EQU7##
[0208] A dose-response curve will be obtained by plotting bound
fluorescence values (mP) against total concentration of the sHLA
molecule on a log-log plot. There should be a plateau effect, with
supraoptimal concentrations of binder yielding no further increase
in mP. Data points are fitted to the dose-response model using the
dose-response equation. Reproducibility can be shown by averaging
the value from 3 independent measurements.
[0209] No binding should be detected in the case of using an
irrelevant fluorescent-labeled MHC molecule. No binding should be
detected in the case of using an irrelevant fluorescent-labeled
peptide.
[0210] Imprecision is the standard deviation of the mean of each
group of mP values. This should generally be less than 10 mP. The
signal to background ratios is determined to assure the quality of
the results. [0211] "Protein+Peptide tracer"/"Protein only"
[0212] A representative value for signal/background can be
calculated from the Parallel (S) value of signal ("Protein+Peptide
tracer") to background ("Protein only") values. Ideally, signal to
background values of at least 10-fold should be targeted.
[0213] The assay range as the change in polarization is calculated
by subtracting the mean mP of ("Free peptide tracer only") from the
mean mP of the ("Protein+Peptide tracer"). Ideally, the net change
in polarization should be greater than 70 mP. Maximum measurable
binding is determined to be the ("Protein+Peptide tracer") values
background subtracted with ("Protein only") controls (maximum sHLA
and tracer binding allowed, which gives a high mP) minus the "Free
peptide tracer only" (minimum tracer binding, which gives a very
low mP). The difference between these two conditions is the maximum
delta mP.
[0214] Each background-corrected mP value obtained is normalized at
a specific sHLA concentration by dividing by the maximum delta mP.
To calculate % of maximum binding, multiply by 100%. Optimal SHLA
concentration is chosen by comparing the results according to their
best signal-to-background ratio and highest polarization value.
[0215] Given milligram to gram quantities of sHLA for the many HLA
alleles in the population, it is possible to automate fluorescent
epitope binding assays with sHLA and thousands to millions of
various peptides. Robotics or other high throughput methods
introduce various peptides to the various sHLA molecules, incubate
at the time and temperatures described herein, and determine those
peptides that bind to a given sHLA molecule and the relative
affinity of those peptides for a sHLA molecule. For example, all
the possible overlapping peptides in 50 different viral pathogens
could be synthesized and tested for their binding to 50 different
sHLA molecules. The resulting data would provide a database of
those viral peptides that bind to HLA molecules for possible
presentation to the immune response. Such data would be useful for
viral vaccine discovery. In a similar test, all peptides encoded by
genes known to be upregulated in tumor cells are tested for their
binding to sHLA molecules. The resulting data would indicate
peptides for potential use in tumor vaccines. Finally, all human
proteins could have overlapping peptides synthesized and tested for
binding to sHLA molecules. The resulting data, combined with gene
expression studies, is useful in determining those epitopes which
are involved in autoimmune disorders. The resulting data could then
be used to design therapies and diagnostics.
Results from Epitope Binding Assay Using Flouorescence
Polarization
[0216] Methods for published peptide binding assays with detergent
lysate HLA or with bacterial produced HLA typically incubate
peptides and HLA at room temperature. FIG. 6 illustrates that
incubating various concentrations of sHLA with a fixed
concentration of peptide at room temperature requires 10,000 to
100,000 picomoles to obtain significant binding of fluorescent
peptide as detected by fluorescent polarization. Baseline levels of
FP are approximately 20 mp while maximum FP at room temperature
incubation is approximately 70; a difference of 50 is the minimum
required to differentiate between a postive and negative binding
peptide.
[0217] By comparison to the incubation of peptide and sHLA at room
temperature, as shown in FIG. 7, it can be seen that heating the
sHLA to 54.degree. Celsius for 45 minutes followed by incubation of
the sHLA with the same peptide at 4.degree. Celsius produces a much
stronger FP signal at the same concentrations of sHLA. The
difference between background and the strongest signal was
approximately 130 mp. A greater difference between the baseline and
the positive allows us to use less sHLA to get a signal difference
of 50 mp and also allows the assay to provide greater
differentiation among the various binding affinities of different
peptides.
[0218] FIG. 8 demonstrates that sHLA molecules bind fluorescent
peptides in a manner specific to the sHLA being tested. In this
Figure, the sHLA molecule A*0201 was tested for its ability to bind
5 peptides specific for A*0201 (+P1 to +P5) and five peptides
specific for the HLA molecule B*2705. The sHLA A*0201 was heated to
54.degree. C. for 45 minutes and then added to a reaction mixture
at 4.degree. C. that contained the various peptides. The data shows
that the sHLA-A*0201 specifically binds A*0201 peptides as detected
by FP.
[0219] FIG. 9 demonstrates that sHLA A*0201 at a concentration of
50 micrograms per milliliter bind peptides specific for A*0201.
Furthermore, this data demonstrates that most peptides specific for
A*0201 bind to their maximum extent within 24 hours. This assay
shows that peptides bind to the sHLA molecules in a specific way
with an FP difference of up to 200 mp. We have therefore identified
reaction conditions that quickly detect a peptide's ability to bind
sHLA molecules. The strong FP difference allows us to compare
peptide binding affinities of various peptides for the sHLA
molecules.
[0220] In the previous figures of this application, we demonstrate
that A*0201 specific peptide ligands bind to sHLA A*0201. In FIG.
10, we add a peptide that is also specific for A*0201 (A2 peptide
(KLGEFYNQMM) (SEQ ID NO:21)) . This "cold" competitor peptide is
mixed with the "hot" fluorescent peptide (peptide P5, having the
sequence ALMDKVLK(FITC)V (SEQ ID NO:22)) at various concentrations
of the cold peptide. The sHLA which has been heated to 54 degrees
Celsius for 45 minutes is added to the mixture of peptides which is
at 4 degrees Celsius. The mixture is then incubated at 4 degrees.
In FIG. 10 it can be seen that increasing concentrations of the
cold peptide prevent the hot peptide from binding to sHLA A*0201.
The less the hot peptide binds, the less FP is obtained. Thus, we
demonstrate that peptides specific for sHLA A*0201 can compete with
labeled peptides specific for A*0201. In this way we can determine
the comparative affinity of various peptides for A*0201: the lower
the concentration needed to compete the hot peptide means that the
cold competitor has a higher affinity.
[0221] Thus, in accordance with the present invention, there has
been provided a method for assaying epitope binding to HLA that
includes methodology for producing and manipulating Class I and
Class II MHC molecules from gDNA as well as methodology for
directing discovering epitopes unique to infected or tumor cells
that fully satisfies the objectives and advantages set forth herein
above. Although the invention has been described in conjunction
with the specific drawings, experimentation, results and language
set forth herein above, it is evident that many alternatives,
modifications, and variations will be apparent to those skilled in
the art. Accordingly, it is intended to embrace all such
alternatives, modifications and variations that fall within the
spirit and broad scope of the invention.
Sequence CWU 1
1
22 1 10 PRT HIV MN-1 1 Glu Gln Met Phe Glu Asp Ile Ile Ser Leu 1 5
10 2 9 PRT Homo sapiens 2 Ile Pro Cys Leu Leu Ile Ser Phe Leu 1 5 3
10 PRT Homo sapiens 3 Ser Thr Thr Ala Ile Cys Ala Thr Gly Leu 1 5
10 4 8 PRT Homo sapiens 4 Ala Pro Ala Gln Asn Pro Glu Leu 1 5 5 9
PRT Homo sapiens 5 Leu Val Met Ala Pro Arg Thr Val Leu 1 5 6 9 PRT
Homo sapiens misc_feature (5)..(5) n or s 6 Ala Pro Phe Ile Xaa Pro
Ala Asp Xaa 1 5 7 9 PRT Homo sapiens 7 Thr Pro Gln Ser Asn Arg Pro
Val Met 1 5 8 9 PRT Homo sapiens 8 Ala Ala Arg Pro Ala Thr Ser Thr
Leu 1 5 9 9 PRT Homo sapiens 9 Met Ala Met Met Ala Ala Leu Met Ala
1 5 10 9 PRT Homo sapiens 10 Ile Ala Thr Val Asp Ser Tyr Val Ile 1
5 11 11 PRT Homo sapiens 11 Ser Pro Asn Gln Ala Arg Ala Gln Ala Ala
Leu 1 5 10 12 10 PRT Homo sapiens 12 Gly Pro Arg Thr Ala Ala Leu
Gly Leu Leu 1 5 10 13 10 PRT Homo sapiens 13 Asn Pro Asn Gln Asn
Lys Asn Val Ala Leu 1 5 10 14 9 PRT Homo sapiens 14 Arg Pro Tyr Ser
Asn Val Ser Asn Leu 1 5 15 9 PRT Homo sapiens 15 Leu Pro Gln Ala
Asn Arg Asp Thr Leu 1 5 16 9 PRT Homo sapiens 16 Gln Pro Arg Tyr
Pro Val Asn Ser Val 1 5 17 8 PRT Homo sapiens 17 Ala Pro Ala Tyr
Ser Arg Ala Leu 1 5 18 9 PRT Homo sapiens 18 Ala Pro Lys Arg Pro
Pro Ser Ala Phe 1 5 19 11 PRT Homo sapiens 19 Ala Ala Ser Lys Glu
Arg Ser Gly Val Ser Leu 1 5 10 20 7 PRT Homo sapiens 20 Gly Ser His
Ser Met Arg Tyr 1 5 21 10 PRT Homo sapiens 21 Lys Leu Gly Glu Phe
Tyr Asn Gln Met Met 1 5 10 22 9 PRT Homo sapiens MISC_FEATURE
(8)..(8) FITC label 22 Ala Leu Met Asp Lys Val Leu Lys Val 1 5
* * * * *